Regulation of FUSCA3 Expression During Seed Development in Arabidopsis.

FUSCA3 (FUS3) is a master regulator of seed development important in establishing and maintaining embryonic identity whose expression is tightly regulated at genetic and epigenetic levels. Despite this prominent role, the control of FUS3 expression remains poorly understood. Promoter and functional complementation analyses provided insight into the regulation of FUS3. W-boxes present in the promoter proximal to the start of transcription are recognized by WRKY type-1 factors which are necessary for the activation of FUS3 expression. The RY motif, the binding site of B3 factors, is important for the activation of FUS3 in the embryo proper but not in the suspensor. The loss of a negative regulatory sequence (NRS) leads to preferential expression of FUS3 in the vasculature of vegetative tissues. Since the NRS includes the RY motif, mechanisms of activation and repression target adjacent or overlapping regions. These findings discriminate the regulation of FUS3 from that of LEAFY COTYLEDON2 by the control exerted by WRKY factors and by the presence of the RY motif, yet also confirm conservation of certain regulatory elements, thereby implicating potential regulation by BASIC PENTACYSTEINE (BPC) factors and POLYCOMB REPRESSIVE COMPLEX2 (PRC2).

Regulation of FATTY ACID ELONGATION1 expression in embryonic and vascular tissues of Brassica napus.

The expression of the FATTY ACID ELONGATION1 genes was characterised to provide insight into the regulation of very long chain fatty acid (VLCFA) biosynthesis in Brassica napus embryos. Each of the two rapeseed homoeologous genes (Bn-FAE1.1 and Bn-FAE1.2) encoding isozymes of 3-keto-acylCoA synthase, a subunit of the cytoplasmic acyl-CoA elongase complex that controls the production of elongated fatty acids, are expressed predominantly in developing seeds. The proximal regions of the Bn-FAE1.1 and Bn-FAE1.2 promoters possess strong sequence identity suggesting that transcriptional control of expression is mediated by this region which contains putative cis-elements characteristic of those found in the promoters of genes expressed in embryo and endosperm. Histochemical staining of rapeseed lines expressing Bn-FAE1.1 promoter:reporter gene fusions revealed a strong expression in the embryo cotyledon and axis throughout the maturation phase. Quantitative analyses revealed the region, -331 to -149, exerts a major control on cotyledon specific expression and the level of expression. A second region, -640 to -475, acts positively to enhance expression levels and extends expression of Bn-FAE1.1 into the axis and hypocotyl but also acts negatively to repress expression in the root meristem. The expression of the Bn-FAE1.1 gene was not restricted to the seed but was also detected in the vascular tissues of germinating seedlings and mature plants in the fascicular cambium tissue present in roots, stem and leaf petiole. We propose that Bn-FAE1.1 expression in vascular tissue may contribute VLCFA for barrier lipid synthesis and reflects the ancestral function of FAE1 encoded 3-keto-acylCoA synthase.

Complementation of Seed Maturation Phenotypes by Ectopic Expression of ABSCISIC ACID INSENSITIVE3, FUSCA3 and LEAFY COTYLEDON2 in Arabidopsis.

ABSCISIC ACID INSENSITIVE3 (ABI3), FUSCA3 (FUS3) and LEAFY COTYLEDON2 (LEC2), collectively the AFL, are master regulators of seed maturation processes. This study examined the role of AFL in the production of seed reserves in Arabidopsis. Quantification of seed reserves and cytological observations of afl mutant embryos show that protein and lipid but not starch reserves are spatially regulated by AFL. Although AFL contribute to a common regulation of reserves, ABI3 exerts a quantitatively greater control over storage protein content whereas FUS3 controls lipid content to a greater extent. Although ABI3 controls the reserve content throughout the embryo, LEC2 and FUS3 regulate reserves in distinct embryonic territories. By analyzing the ability of an individual ectopically expressed AFL to suppress afl phenotypes genetically, we show that conserved domains common to each component of the AFL are sufficient for the initiation of storage product synthesis and the establishment of embryo morphology. This confirms redundancy among the AFL and indicates a threshold necessary for function within the AFL pool. Since no individual AFL was able to suppress the tolerance to desiccation, mid- and late-maturation programs were uncoupled.

Chromodomain, Helicase and DNA-binding CHD1 protein, CHR5, are involved in establishing active chromatin state of seed maturation genes.

Chromatin modification and remodelling are the basis for epigenetic regulation of gene expression. LEAFY COTYLEDON 1 (LEC1), LEAFY COTYLEDON 2 (LEC2), ABSCISIC ACID-INSENSITIVE 3 (ABI3) and FUSCA3 (FUS3) are key regulators of embryo development and are repressed after seed maturation. The chromatin remodelling CHD3 protein PICKLE (PKL) is involved in the epigenetic silencing of the genes. However, the chromatin mechanism that establishes the active state of these genes during early embryo development is not clear. We show that the Arabidopsis CHD1-related gene, CHR5, is activated during embryo development. Mutation of the gene reduced expression of LEC1, ABI3 and FUS3 in developing embryo and accumulation of seed storage proteins. Analysis of double mutants revealed an antagonistic function between CHR5 and PKL in embryo gene expression and seed storage protein accumulation, which likely acted on the promoter region of the genes. CHR5 was shown to be associated with the promoters of ABI3 and FUS3 and to be required to reduce nucleosome occupancy near the transcriptional start site. The results suggest that CHR5 is involved in establishing the active state of embryo regulatory genes by reducing nucleosomal barrier, which may be exploited to enhance seed protein production.

ATR3 encodes a diflavin reductase essential for Arabidopsis embryo development.

*The Arabidopsis genome possesses two confirmed Cytochrome P450 Reductase (CPR) genes, ATR1 and ATR2, together with a third putative homologue, ATR3, which annotation is questionable. *Phylogenetic analysis classified ATR3 as a CPR-like protein sharing homologies with the animal cytosolic dual flavin reductases, NR1 and Fre-1, distinct from the microsomal CPRs, ATR1 and ATR2. Like NR1 and Fre-1, ATR3 lacks the N-terminal endoplasmic reticulum (ER) anchor domain of CPRs and is localized in the cytoplasm. Recombinant ATR3 in plant soluble extracts was able to reduce cytochrome c but failed to reduce the human P450 CYP1A2. *Loss of ATR3 function resulted in early embryo lethality indicating that this reductase activity is essential. A yeast 2-hybrid screen identified a unique interaction of ATR3 with the homologue of the human anti-apoptotic CIAPIN1 and the yeast Dre2 protein. *This interaction suggests two possible roles for ATR3 in the control of cell death and in chromosome segregation at mitosis. Consistent with these results, the promoter of ATR3 is activated during cell cycle progression. Together these results demonstrated that ATR3 belongs to the NR1 subfamily of diflavin reductases whose characterized members are involved in essential cellular functions.

The first zygotic division in Arabidopsis requires de novo transcription of thymidylate kinase.

Re-activation of cell division after fertilization involves the specific regulation of a set of genes. To identify genes involved in the gametophytic to sporophytic transition, we screened Arabidopsis T-DNA insertion lines for early seed abortion at the zygote (zeus) or one-cell embryo stages (cyclops), and characterized a sporophytic zygote-lethal mutation, zeus1. ZEUS1 encodes a thymidylate kinase (AtTMPK) that synthesizes dTDP and is involved in the regulation of DNA replication. Unlike in yeast and animals, the single AtTMPK gene is capable of producing two proteins by alternative splicing; the longer isoform is targeted to the mitochondria, the shorter to the cytosol. Transcription of AtTMPK is activated during the G(1)/S-phase transition of the cell cycle, similarly to yeast and mammalian orthologues. In AtTMPK:GUS plants, the reporter gene was preferentially expressed in cells undergoing division, but was not detected during the male and female gametophytic mitoses. GUS expression was observed in mature embryo sacs prior to fertilization, and this expression may indicate the time of synchronization of the gamete cell-cycle phases. Identification of ZEU1 emphasizes the importance of control of the metabolism of DNA in the regulation of the G(1)/S-phase transition at fertilization.

HvPG1 and ECA1: two genes activated transcriptionally in the transition of barley microspores from the gametophytic to the embryogenic pathway.

Microspores genetically programmed to produce male gametes can be switched to the embryogenic pathway to give rise to haploid embryos. Microspore embryogenesis is usually induced in barley by stress pre-treatment applied to vacuolated microspores. We studied the expression of two genes during the early stages of microspore embryogenesis to gain further insight into the microspore transition from the gametophytic to the embryogenic pathway. RT-PCR together with in situ hybridization on sections (ISH) and whole-mount in situ hybridization (WISH) were used to analyse the expression of the early-culture abundant gene (ECA1), which is expressed in barley during microspore embryogenesis, and a polygalacturonase gene (HvPG1), a late pollen gene expressed during gametogenesis only after microspore division. Both ECA1 and HvPG1 genes were transcriptionally active after stress pre-treatment in the same populations of microspore-derived structures, representing the sporophytically induced ones. ECA1 transcripts were also detected after 3 days' culture. Our results point to the possibility of using ECA1 gene expression as a marker for the induction of microspore embryogenesis and the earliest stages of this process. Finally, we demonstrate that WISH is a suitable technique for studying gene expression in embryogenic microspore populations and, because different structures can be examined individually, is an appropriate complement to transcriptomic profile analyses in the study of early microspore embryogenesis.

A bona fide La protein is required for embryogenesis in Arabidopsis thaliana.

Searches in the Arabidopsis thaliana genome using the La motif as query revealed the presence of eight La or La-like proteins. Using structural and phylogenetic criteria, we identified two putative genuine La proteins (At32 and At79) and showed that both are expressed throughout plant development but at different levels and under different regulatory conditions. At32, but not At79, restores Saccharomyces cerevisiae La nuclear functions in non-coding RNAs biogenesis and is able to bind to plant 3'-UUU-OH RNAs. We conclude that these La nuclear functions are conserved in Arabidopsis and supported by At32, which we renamed as AtLa1. Consistently, AtLa1 is predominantly localized to the plant nucleoplasm and was also detected in the nucleolar cavity. The inactivation of AtLa1 in Arabidopsis leads to an embryonic-lethal phenotype with deficient embryos arrested at early globular stage of development. In addition, mutant embryonic cells display a nucleolar hypertrophy suggesting that AtLa1 is required for normal ribosome biogenesis. The identification of two distantly related proteins with all structural characteristics of genuine La proteins suggests that these factors evolved to a certain level of specialization in plants. This unprecedented situation provides a unique opportunity to dissect the very different aspects of this crucial cellular activity.

The alpha-N-acetyl-glucosaminidase gene is transcriptionally activated in male and female gametes prior to fertilization and is essential for seed development in Arabidopsis.

Sugar residues in proteoglycan complexes carry important signalling and regulatory functions in biology. In humans, heparan sulphate is an example of such a complex polymer containing glucosamine and N-acetyl-glucosamine residues and is present in the extracellular matrix. Although heparan sulphate has not been found in plants, the At5g13690 gene encoding the alpha-N-acetyl-glucosaminidase (NAGLU), an enzyme involved in its catabolism, is present in the Arabidopsis genome. Among our collection of embryo-defective lines, a plant was identified in which the T-DNA had inserted into the AtNAGLU gene. The phenotype of atnaglu is an early arrest of seed development without apparent male or female gametophytic effects. These data demonstrated the essential function in Arabidopsis consistent with the contribution of NAGLU to the Sanfilippo syndrome in human. Expression of AtNAGLU in plants was shown to be prevalent during reproductive development. The presence of AtNAGLU mRNA was observed during early and late male gametogenesis and in each cell of the embryo sac at the time of fertilization. After fertilization, AtNAGLU was expressed in the embryo, suspensor, and endosperm until the cotyledonary stage embryo. This precise pattern of expression identifies the cells and tissues where a remodelling of the N-acetyl-glucosamine residues of proteoglycan complexes is occurring. This work provides original evidence of the important role of N-acetyl-glucosamines in plant reproductive development.

The importance of being essential: EMBRYO-DEFECTIVE genes in Arabidopsis.

With the completion of the sequence of the first bacterial genomes, scientists have been able to address the question: How many genes are required for cell viability? In attempting to reply to this question, the concept of the minimal gene set was developed and validated by systematic gene disruption. In a similar manner, whole genome comparisons and systematic Knock-Out have been performed in eukaryotes and have led to the identification to date of the set of essential genes in yeast and C. elegans. In the plant kingdom, the sequence of the Arabidopsis genome together with large-scale functional genomics programs now allow us to address the question of essentiality in Arabidopsis. These concerted efforts have resulted in the identification to date of up to 219 genes essential for seed development (EMBRYO-DEFECTIVE, EMB, genes). With this basic knowledge, we can start a valid comparison of essentiality in Arabidopsis and in other eukaryotes based on functional categories and orthologous relationships. Furthermore, the function of the EMB genes in the particular context of eukaryote evolution driven by whole genome duplications and selective gene loss will be discussed.

E2F factors rate controls the dual role of CDE/E2F composite element: a model of E2F-regulated gene expression in plant development.

The promoters of several E2F-regulated genes identified in plants contain a variety of E2F motifs, notably a composite element consisting of a "CDE-like element" C/GGCGG on one strand, described as repressor in animals, associated with an E2F element on the complementary strand. This detailed study throughout plant development using ribonucleotide reductase promoters, allows us to propose a model, where E2F and composite elements play a dual role. Such regulation is mainly conditioned by the availability of E2F factors in tissues and during the cell cycle in tobacco.

Seed maturation: Simplification of control networks in plants.

Networks controlling developmental or metabolic processes in plants are often complex as a consequence of the duplication and specialisation of the regulatory genes as well as the numerous levels of transcriptional and post-transcriptional controls added during evolution. Networks serve to accommodate multicellular complexity and increase robustness to environmental changes. Mathematical simplification by regrouping genes or pathways in a limited number of hubs has facilitated the construction of models for complex traits. In a complementary approach, a biological simplification can be achieved by using genetic modification to understand the core and singular ancestral function of the network, which is likely to be more prevalent within the plant kingdom rather than specific to a species. With this viewpoint, we review examples of simplification successfully undertaken in yeast and other organisms. A strategy of progressive complementation of single, double and triple mutants of seed maturation confirmed the fundamental role of the AFL sub-family of B3 transcription factors as master regulators of seed maturation, illustrating that biological simplification of complex networks could be more widely applied in plants. Defining minimal control networks will facilitate evolutionary comparisons of regulatory processes and the identification of an essential gene set for synthetic biology.

Flanking sequence tags in Arabidopsis thaliana T-DNA insertion lines: a pilot study.

Eight hundred and fifty Arabidopsis thaliana T-DNA insertion lines have been selected on a phenotypic basis. The T-DNA flanking sequences (FST) have been isolated using a PCR amplification procedure and sequenced. Seven hundred plant DNA sequences have been obtained revealing a T-DNA insertion in, or in the immediate vicinity of 482 annotated genes. Limited deletions of plant DNA have been observed at the site of insertion of T-DNA as well as in its left (LB) and right (RB) T-DNA signal sequences. The distribution of the T-DNA insertions along the chromosomes shows that they are essentially absent from the centrometric and pericentrometric regions.

Redrawing the borderline: Control of DNA replication at fertilization.

In our recent paper in Plant Journal, we described the transcriptional activation of AtTMPK (thymidylate kinase),1 a recognized G(1)/S phase marker of the cell cycle progression and its role in early seed development. Here, we compare our conclusions on the regulation of AtTMPK and those of other genes participating in DNA replication, including DPB2, a subunit of the DNA polymerase epsilon complex.2 Although, the dual localisation of AtTMPK in the cytosol and mitochondria seems to be unique to plants, this phenomenon of multiple targeting is also used for other proteins involved in DNA replication, such as DNA ligase 1 (AtLIG1), and may represent a way to coordinate nuclear and organellar divisions.

Characterization of leafy cotyledon1-like during embryogenesis in Theobroma cacao L.

Theobroma cacao L., an economically important crop for developing countries, can be experimentally propagated by somatic embryogenesis. Because of their potential roles in embryogenesis, a gene candidate strategy was initiated to find gene homologues of the members of the leafy cotyledon family of transcription factors. A homologue of the leafy cotyledon1-like gene, that encodes the HAP 3 subunit of the CCAAT box-binding factor, was found in the cocoa genome (TcL1L). The translated peptide shared a high amino acid sequence identity with the homologous genes of Arabidopsis thaliana, Phaseolus coccineus and Helianthus annuus. TcL1L transcripts mainly accumulated in young and immature zygotic embryos, and, to a lesser extent, in young and immature somatic embryos. In situ hybridization specified the localization of the transcripts as being mainly in embryonic cells of young embryos, the meristematic cells of the shoot and root apex of immature embryos, and in the protoderm and epidermis of young and immature embryos, either zygotic or somatic. Non-embryogenic explants did not show TcL1L expression. Ectopic expression of the TcL1L gene could partially rescue the Arabidopsis lec1 mutant phenotype, suggesting a similarity of function in zygotic embryogenesis.

QQT proteins colocalize with microtubules and are essential for early embryo development in Arabidopsis.

During Arabidopsis embryogenesis, the control of division between daughter cells is critical for pattern formation. Two embryo-defective (emb) mutant lines named quatre-quart (qqt) were characterized by forward and reverse genetics. The terminal arrest of qqt1 and qqt2 embryos was at the octant stage, just prior to the round of periclinal divisions that establishes the dermatogen stage . Homozygous embryos of a weaker allele of qqt1 were able to divide further, resulting in aberrant periclinal divisions. These phenotypic analyses support an essential role of the QQT proteins in the correct formation of the tangential divisions. That an important proportion of qqt1 embryos were arrested prior to the octant stage indicated a more general role in cell division. The analysis of QQT1 and QQT2 genes revealed that they belong to a small subgroup of the large family encoding ATP/GTP binding proteins, and are widely conserved among plants, vertebrates and Archaea. We showed that QQT1 and QQT2 proteins interact with each other in a yeast two-hybrid system, and that QQT1 and QQT2 tagged by distinct fluorescent probes colocalize with microtubules during mitosis, in agreement with their potential role in cell division and their mutant phenotype. We propose that QQT1 and QQT2 proteins participate in the organization of microtubules during cell division, and that this function is essential for the correct development of the early embryo.

A network of local and redundant gene regulation governs Arabidopsis seed maturation.

In Arabidopsis thaliana, four major regulators (ABSCISIC ACID INSENSITIVE3 [ABI3], FUSCA3 [FUS3], LEAFY COTYLEDON1 [LEC1], and LEC2) control most aspects of seed maturation, such as accumulation of storage compounds, cotyledon identity, acquisition of desiccation tolerance, and dormancy. The molecular basis for complex genetic interactions among these regulators is poorly understood. By analyzing ABI3 and FUS3 expression in various single, double, and triple maturation mutants, we have identified multiple regulatory links among all four genes. We found that one of the major roles of LEC2 was to upregulate FUS3 and ABI3. The lec2 mutation is responsible for a dramatic decrease in ABI3 and FUS3 expression, and most lec2 phenotypes can be rescued by ABI3 or FUS3 constitutive expression. In addition, ABI3 and FUS3 positively regulate themselves and each other, thereby forming feedback loops essential for their sustained and uniform expression in the embryo. Finally, LEC1 also positively regulates ABI3 and FUS3 in the cotyledons. Most of the genetic controls discovered were found to be local and redundant, explaining why they had previously been overlooked. This works establishes a genetic framework for seed maturation, organizing the key regulators of this process into a hierarchical network. In addition, it offers a molecular explanation for the puzzling variable features of lec2 mutant embryos.

Hordeins are expressed in microspore-derived embryos and also during male gametophytic and very early stages of seed development.

Microspore-derived embryos induced by anther or isolated-microspore culture display certain characteristics of zygotic embryos. Furthermore, the expression of certain endosperm genes has been described in these non-zygotic embryos. The expression of hordein genes encoding the main barley endosperm proteins has been studied using a wide range of methods (RT-PCR, in situ hybridization, ELISA sandwich, western blotting immunocytochemistry, and cytochemistry) to ascertain their presence or absence during the induction and first stages of microspore embryogenesis. Due to the very sensitive techniques used it was possible to detect for the first time hordein expression during microspore embryogenesis. Surprisingly, these hordeins were also detected at different stages of male gametophytic development as well as during the very early stages of seed development, when they have not hitherto been detected. The expression and localization of these storage proteins and their corresponding transcripts provide new information about barley microspore embryogenesis and its relationship to zygotic embryogenesis. Although only small quantities of hordeins are accumulated during microspore embryogenesis they seem to be necessary for the initial development of the microspore-derived embryo. This idea is supported by the changes detected in their concentration throughout this process and is in accordance with previously published data concerning the importance of endosperm proteins for embryo development in both microspore culture and in planta.

Genetic analysis of two Arabidopsis DNA polymerase epsilon subunits during early embryogenesis.

Accurate DNA replication is one of the most important events in the life of a cell. To perform this task, the cell utilizes several DNA polymerase complexes. We investigated the role of DNA polymerase epsilon during gametophyte and seed development using forward and reverse genetic approaches. In Arabidopsis, the catalytic subunit of this complex is encoded by two genes, AtPOL2a and AtPOL2b, whereas the second largest regulatory subunit AtDPB2 is present as a unique complete copy. Disruption of AtPOL2a or AtDPB2 resulted in a sporophytic embryo-defective phenotype, whilst mutations in AtPOL2b produced no visible effects. Loss of AtDPB2 function resulted in a severe reduction in nuclear divisions, both in the embryo and in the endosperm. Mutations in AtPOL2a allowed several rounds of mitosis to proceed, often with aberrant planes of division. Moreover, AtDPB2 was not expressed during development of the female gametophyte, which requires three post-meiotic nuclear divisions. Since a consensus binding site for E2F transcription factors was identified in the promoter region of both genes, the promoter-reporter fusion technique was used to show that luciferase activity was increased at specific phases of the cell cycle in synchronized tobacco BY-2 cells. Our results support the idea that fertilization may utilize the mechanisms of cell cycle transcriptional regulation of genes to reactivate the divisions of the oosphere and central cell.

Proanthocyanidin-accumulating cells in Arabidopsis testa: regulation of differentiation and role in seed development.

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.