Molecular and epigenetic regulations and functions of the LAFL transcriptional regulators that control seed development.

The LAFL (i.e. LEC1, ABI3, FUS3, and LEC2) master transcriptional regulators interact to form different complexes that induce embryo development and maturation, and inhibit seed germination and vegetative growth in Arabidopsis. Orthologous genes involved in similar regulatory processes have been described in various angiosperms including important crop species. Consistent with a prominent role of the LAFL regulators in triggering and maintaining embryonic cell fate, their expression appears finely tuned in different tissues during seed development and tightly repressed in vegetative tissues by a surprisingly high number of genetic and epigenetic factors. Partial functional redundancies and intricate feedback regulations of the LAFL have hampered the elucidation of the underpinning molecular mechanisms. Nevertheless, genetic, genomic, cellular, molecular, and biochemical analyses implemented during the last years have greatly improved our knowledge of the LALF network. Here we summarize and discuss recent progress, together with current issues required to gain a comprehensive insight into the network, including the emerging function of LEC1 and possibly LEC2 as pioneer transcription factors.

LEC1 (NF-YB9) directly interacts with LEC2 to control gene expression in seed.

The LAFL transcription factors LEC2, ABI3, FUS3 and LEC1 are master regulators of seed development. LEC2, ABI3 and FUS3 are closely related proteins that contain a B3-type DNA binding domain. We have previously shown that LEC1 (a NF-YB type protein) can increase LEC2 and ABI3 but not FUS3 activity. Interestingly, FUS3, LEC2 and ABI3 contain a B2 domain, the function of which remains elusive. We showed that LEC1 and LEC2 partially co-localised in the nucleus of developing embryos. By comparing protein sequences from various species, we identified within the B2 domains a set of highly conserved residues (i.e. TKxxARxxRxxAxxR). This domain directly interacts with LEC1 in yeast. Mutations of the conserved amino acids of the motif in the B2 domain abolished this interaction both in yeast and in moss protoplasts and did not alter the nuclear localisation of LEC2 in planta. Conversely, the mutations of key amino acids for the function of LEC1 in planta (D86K) prevented the interaction with LEC2. These results provide molecular evidences for the binding of LEC1 to B2-domain containing transcription factors, to form heteromers, involved in the control of gene expression.

Profiling the onset of somatic embryogenesis in Arabidopsis.

BACKGROUND: Totipotency is the ability of a cell to regenerate a whole organism. Plant somatic embryogenesis (SE) is a remarkable example of totipotency because somatic cells reverse differentiation, respond to an appropriate stimulus and initiate embryo development. Although SE is an ideal system to investigate de-differentiation and differentiation, we still lack a deep molecular understanding of the phenomenon due to experimental restraints. RESULTS: We applied the INTACT method to specifically isolate the nuclei of those cells undergoing SE among the majority of non-embryogenic cells that make up a callus. We compared the transcriptome of embryogenic cells to the one of proliferating callus cells. Our analyses revealed that embryogenic cells are transcriptionally rather than metabolically active. Embryogenic cells shut off biochemical pathways involved in carbohydrate and lipid metabolism and activate the transcriptional machinery. Furthermore, we show how early in SE, ground tissue and leaf primordia specification are switched on before the specification of a shoot apical meristem. CONCLUSIONS: This is the first attempt to specifically profile embryogenic cells among the different cell types that constitute plant in vitro tissue cultures. Our comparative analyses provide insights in the gene networks regulating SE and open new research avenues in the field of plant regeneration.

Regulation and evolution of the interaction of the seed B3 transcription factors with NF-Y subunits.

The LAFL genes (LEC2, ABI3, FUS3, LEC1) encode transcription factors that regulate different aspects of seed development, from early to late embryogenesis and accumulation of storage compounds. These transcription factors form a complex network, with members able to interact with various other players to control the switch between embryo development and seed maturation and, at a later stage in the plant life cycle, between the mature seed and germination. In this review, we first summarize our current understanding of the role of each member in the network in the light of recent advances regarding their regulation and structure/function relationships. In a second part, we discuss new insights concerning the evolution of the LAFL genes to address the more specific question of the conservation of LEAFY COTYLEDONS 2 in both dicots and monocots and the putative origin of the network. Last we examine the current major limitations to current knowledge and future prospects to improve our understanding of this regulatory network.

TRANSPARENT TESTA 16 and 15 act through different mechanisms to control proanthocyanidin accumulation in Arabidopsis testa.

Flavonoids are secondary metabolites that fulfil a multitude of functions during the plant life cycle. In Arabidopsis proanthocyanidins (PAs) are flavonoids that specifically accumulate in the innermost integuments of the seed testa (i.e. endothelium), as well as in the chalaza and micropyle areas, and play a vital role in protecting the embryo against various biotic and abiotic stresses. PAs accumulation in the endothelium requires the activity of the MADS box transcription factor TRANSPARENT TESTA (TT) 16 (ARABIDOPSIS B-SISTER/AGAMOUS-LIKE 32) and the UDP-glycosyltransferase TT15 (UGT80B1). Interestingly tt16 and tt15 mutants display a very similar flavonoid profiles and patterns of PA accumulation. By using a combination of genetic, molecular, biochemical, and histochemical methods, we showed that both TT16 and TT15 act upstream the PA biosynthetic pathway, but through two distinct genetic routes. We also demonstrated that the activity of TT16 in regulating cell fate determination and PA accumulation in the endothelium is required in the chalaza prior to the globular stage of embryo development. Finally this study provides new insight showing that TT16 and TT15 functions extend beyond PA biosynthesis in the inner integuments of the Arabidopsis seed coat.

A new system for fast and quantitative analysis of heterologous gene expression in plants.

• Large-scale analysis of transcription factor-cis-acting element interactions in plants, or the dissection of complex transcriptional regulatory mechanisms, requires rapid, robust and reliable systems for the quantification of gene expression. • Here, we describe a new system for transient expression analysis of transcription factors, which takes advantage of the fast and easy production and transfection of Physcomitrella patens protoplasts, coupled to flow cytometry quantification of a fluorescent protein (green fluorescent protein). Two small-sized and high-copy Gateway® vectors were specifically designed, although standard binary vectors can also be employed. • As a proof of concept, the regulation of BANYULS (BAN), a key structural gene involved in proanthocyanidin biosynthesis in Arabidopsis thaliana seeds, was used. In P. patens, BAN expression is activated by a complex composed of three proteins (TT2/AtMYB123, TT8/bHLH042 and TTG1), and is inhibited by MYBL2, a transcriptional repressor, as in Arabidopsis. Using this approach, two new regulatory sequences that are necessary and sufficient for specific BAN expression in proanthocyanidin-accumulating cells were identified. • This one hybrid-like plant system was successfully employed to quantitatively assess the transcriptional activity of four regulatory proteins, and to identify their target recognition sites on the BAN promoter.

A comprehensive overview of grain development in Brachypodium distachyon variety Bd21.

A detailed and comprehensive understanding of seed reserve accumulation is of great importance for agriculture and crop improvement strategies. This work is part of a research programme aimed at using Brachypodium distachyon as a model plant for cereal grain development and filling. The focus was on the Bd21-3 accession, gathering morphological, cytological, and biochemical data, including protein, lipid, sugars, starch, and cell-wall analyses during grain development. This study highlighted the existence of three main developmental phases in Brachypodium caryopsis and provided an extensive description of Brachypodium grain development. In the first phase, namely morphogenesis, the embryo developed rapidly reaching its final morphology about 18 d after fertilization (DAF). Over the same period the endosperm enlarged, finally to occupy 80% of the grain volume. During the maturation phase, carbohydrates were continuously stored, mainly in the endosperm, switching from sucrose to starch accumulation. Large quantities of ß-glucans accumulated in the endosperm with local variations in the deposition pattern. Interestingly, new ß-glucans were found in Brachypodium compared with other cereals. Proteins (i.e. globulins and prolamins) were found in large quantities from 15 DAF onwards. These proteins were stored in two different sub-cellular structures which are also found in rice, but are unusual for the Pooideae. During the late stage of development, the grain desiccated while the dry matter remained fairly constant. Brachypodium exhibits some significant differences with domesticated cereals. Beta-glucan accumulates during grain development and this cell wall polysaccharide is the main storage carbohydrate at the expense of starch.

At5g50600 encodes a member of the short-chain dehydrogenase reductase superfamily with 11beta- and 17beta-hydroxysteroid dehydrogenase activities associated with Arabidopsis thaliana seed oil bodies.

In a previous work, we presented evidence for the presence of a protein encoded by At5g50600 in oil bodies (OBs) from Arabidopsis thaliana [P. Jolivet, E. Roux, S. D'Andrea, M. Davanture, L. Negroni, M. Zivy, T. Chardot, Protein composition of oil bodies in Arabidopsis thaliana ecotype WS, Plant Physiol. Biochem. 42 (2004) 501-509]. Using specific antibodies and proteomic techniques, we presently confirm the existence of this protein, which is a member of the short-chain steroid dehydrogenase reductase superfamily. We have measured its activity toward various steroids (cholesterol, dehydroepiandrosterone, cortisol, corticosterone, estradiol, estrone) and NAD(P)(H), either within purified OBs or as a purified bacterially expressed chimera. Both enzymatic systems (OBs purified from A. thaliana seeds as well as the chimeric enzyme) exhibited hydroxysteroid dehydrogenase (HSD) activity toward estradiol (17beta-hydroxysteroid) with NAD+ or NADP+, NADP+ being the preferred cofactor. Low levels of activity were observed with cortisol or corticosterone (11beta-hydroxysteroids), but neither cholesterol nor DHEA (3beta-hydroxysteroids) were substrates, whatever the cofactor used. Similar activity profiles were found for both enzyme sources. Purified OBs were found to be also able to catalyze estrone reduction (17beta-ketosteroid reductase activity) with NADPH. The enzyme occurring in A. thaliana OBs can be classified as a NADP+-dependent 11beta-,17beta-hydroxysteroid dehydrogenase/17beta-ketosteroid reductase. This enzyme probably corresponds to AtHSD1, which is encoded by At5g50600. However, its physiological role and substrates still remain to be determined.

FLAGdb/FST: a database of mapped flanking insertion sites (FSTs) of Arabidopsis thaliana T-DNA transformants.

A large collection of T-DNA insertion transformants of Arabidopsis thaliana has been generated at the Institute of Agronomic Research, Versailles, France. The molecular characterisation of the insertion sites is currently performed by sequencing genomic regions flanking the inserted T-DNA (FST). The almost complete sequence of the nuclear genome of A.thaliana provides the framework for organising FSTs in a genome oriented database, FLAGdb/FST (http://genoplante-info.infobiogen.fr). The main scope of FLAGdb/FST is to help biologists to find the FSTs that interrupt the genes in which they are interested. FSTs are anchored to the genome sequences of A.thaliana and positions of both predicted genes and FSTs are shown graphically on sequences. Requests to locate the genomic position of a query sequence are made using BLAST programs. The response delivered by FLAGdb/FST is a graphical representation of the putative FSTs and of predicted genes in a 20 kb region.

LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development.

The Arabidopsis LEAFY COTYLEDON2 (LEC2) gene is a central embryonic regulator that serves critical roles both early and late during embryo development. LEC2 is required for the maintenance of suspensor morphology, specification of cotyledon identity, progression through the maturation phase, and suppression of premature germination. We cloned the LEC2 gene on the basis of its chromosomal position and showed that the predicted polypeptide contains a B3 domain, a DNA-binding motif unique to plants that is characteristic of several transcription factors. We showed that LEC2 RNA accumulates primarily during seed development, consistent with our finding that LEC2 shares greatest similarity with the B3 domain transcription factors that act primarily in developing seeds, VIVIPAROUS1/ABA INSENSITIVE3 and FUSCA3. Ectopic, postembryonic expression of LEC2 in transgenic plants induces the formation of somatic embryos and other organ-like structures and often confers embryonic characteristics to seedlings. Together, these results suggest that LEC2 is a transcriptional regulator that establishes a cellular environment sufficient to initiate embryo development.

The Arabidopsis TT2 gene encodes an R2R3 MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed.

In Arabidopsis, proanthocyanidins specifically accumulate in the endothelium during early seed development. At least three TRANSPARENT TESTA (TT) genes, TT2, TT8, and TTG1, are necessary for the normal expression of several flavonoid structural genes in immature seed, such as DIHYDROFLAVONOL-4-REDUCTASE and BANYULS (BAN). TT8 and TTG1 were characterized recently and found to code for a basic helix-loop-helix domain transcription factor and a WD-repeat-containing protein, respectively. Here the molecular cloning of the TT2 gene was achieved by T-DNA tagging. TT2 encoded an R2R3 MYB domain protein with high similarity to the rice OsMYB3 protein and the maize COLORLESS1 factor. A TT2-green fluorescent protein fusion protein was located mostly in the nucleus, in agreement with the regulatory function of the native TT2 protein. TT2 expression was restricted to the seed during early embryogenesis, consistent with BAN expression and the proanthocyanidin deposition profile. Finally, in gain-of-function experiments, TT2 was able to induce ectopic expression of BAN in young seedlings and roots in the presence of a functional TT8 protein. Therefore, our results strongly suggest that stringent spatial and temporal BAN expression, and thus proanthocyanidin accumulation, are determined at least partially by TT2.

Arabidopsis glucosidase I mutants reveal a critical role of N-glycan trimming in seed development.

Glycoproteins with asparagine-linked (N-linked) glycans occur in all eukaryotic cells. The function of their glycan moieties is one of the central problems in contemporary cell biology. N-glycosylation may modify physicochemical and biological protein properties such as conformation, degradation, intracellular sorting or secretion. We have isolated and characterized two allelic Arabidopsis mutants, gcs1-1 and gcs1-2, which produce abnormal shrunken seeds, blocked at the heart stage of development. The mutant seeds accumulate a low level of storage proteins, have no typical protein bodies, display abnormal cell enlargement and show occasional cell wall disruptions. The mutated gene has been cloned by T-DNA tagging. It codes for a protein homologous to animal and yeast alpha-glucosidase I, an enzyme that controls the first committed step for N-glycan trimming. Biochemical analyses have confirmed that trimming of the alpha1,2- linked glucosyl residue constitutive of the N-glycan precursor is blocked in this mutant. These results demonstrate the importance of N-glycan trimming for the accumulation of seed storage proteins, the formation of protein bodies, cell differentiation and embryo development.

The TT8 gene encodes a basic helix-loop-helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques.

The TRANSPARENT TESTA8 (TT8) locus is involved in the regulation of flavonoid biosynthesis in Arabidopsis. The tt8-3 allele was isolated from a T-DNA-mutagenized Arabidopsis collection and found to be tagged by an integrative molecule, thus permitting the cloning and sequencing of the TT8 gene. TT8 identity was confirmed by complementation of tt8-3 and sequence analysis of an additional allele. The TT8 gene encodes a protein that displays a basic helix-loop-helix at its C terminus and represents an Arabidopsis ortholog of the maize R transcription factors. The TT8 transcript is present in developing siliques and in young seedlings. The TT8 protein is required for normal expression of two flavonoid late biosynthetic genes, namely, DIHYDROFLAVONOL 4-REDUCTASE (DFR) and BANYULS (BAN), in Arabidopsis siliques. Interestingly, TRANSPARENT TESTA GLABRA1 (TTG1) and TT2 genes also control the expression of DFR and BAN genes. Our results suggest that the TT8, TTG1, and TT2 proteins may interact to control flavonoid metabolism in the Arabidopsis seed coat.

The Arabidopsis AtEPR1 extensin-like gene is specifically expressed in endosperm during seed germination.

Screening of 10 000 Arabidopsis transgenic lines carrying a gene-trap (GUS) construct has been undertaken to identify markers of seed germination. One of these lines showed GUS activity restricted to the endosperm, at the micropylar end of the germinating seed. The genomic DNA flanking the T-DNA insert was cloned by walking PCR and the insertion was shown to be located 70 bp upstream of a 2285 bp open reading frame (AtEPR1) sharing strong similarities with extensins. The AtEPR1 open reading frame consists of 40 proline-rich repeats and is expressed in both wild-type and mutant lines. The expression of the AtEPR1 gene appears to be under positive control of gibberellic acid, but is not downregulated by abscisic acid during seed germination. No expression was detected in organs other than endosperm during seed germination. The putative role of AtEPR1 is discussed in the light of its specific expression in relation to seed germination.

The TAG1 locus of Arabidopsis encodes for a diacylglycerol acyltransferase.

Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) is a membrane enzyme that drives the final step in the formation of oils using diacylglycerol (DAG) and acyl-CoA to yield triacylglycerol (TAG). We identified a putative plant DGAT gene (TRIACYLGLYCEROL1: TAG1) and demonstrated its function by the cloning of two mutated alleles, designated AS11 (tag1-1) and ABX45 (tag1-2). One allele, AS11, has been previously characterised at the biochemical level. Mutant seeds contained less oil with a modified fatty acid profile and have reduced germination rates compared to wild-type controls. The TAG1 cDNA encodes for a 520-aa protein that possesses multiple putative transmembrane domains and shows 70 % similarity to a human DGAT cDNA.

Characterization of an Arabidopsis thaliana cDNA homologue to animal poly(ADP-ribose) polymerase.

A full-length Arabidopsis thaliana cDNA (app) encoding a protein with high similarity (about 60%) to the catalytic domain of vertebrate poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) has been cloned. The N-terminal extension of the Arabidopsis protein shows similarities with domains of different nuclear and DNA binding proteins in agreement with nuclear localization and putative function of a plant PARP. APP is encoded by a single gene mapped at the top of chromosome 4 of the Arabidopsis genome and mRNA is abundant in cell suspension culture compared to its accumulation in whole plant.

Kinetic analysis of the non-phosphorylated, in vitro phosphorylated, and phosphorylation-site-mutant (Asp8) forms of intact recombinant C4 phosphoenolpyruvate carboxylase from sorghum.

Steady-state kinetic analyses were performed on the non-phosphorylated, in vitro phosphorylated and phosphorylation-site mutant (Ser8-->Asp) forms of purified recombinant sorghum C4 phosphoenolpyruvate (P-pyruvate) carboxylase (EC 4.1.1.31) containing an intact N-terminus. Significant differences in certain kinetic parameters were observed between these three enzyme forms when activity was assayed at a suboptimal but near-physiological pH (7.3), but not at optimal pH (8.0). Most notably, at pH 7.3 the apparent Ki for the negative allosteric effector L-malate was 0.17 mM, 1.2 mM and 0.45 mM while the apparent Ka for the positive allosteric effector glucose 6-phosphate (Glc6P) at 1 mM P-pyruvate was 1.3 mM, 0.28 mM and 0.45 mM for the dephosphorylated, phosphorylated and mutant forms of the enzyme, respectively. These and related kinetic analyses at pH 7.3 show that phosphorylation of C4 P-pyruvate carboxylase near its N-terminus has a relatively minor effect on V and Km (total P-pyruvate) but has a dramatic effect on the extent of activation by Glc6P, type of inhibition by L-malate and, most especially, Ka (Glc6P) and Ki (L-malate). Thus, regulatory phosphorylation profoundly influences the interactive allosteric properties of this cytosolic C4-photosynthesis enzyme.

Purification and characterization of pea thioredoxin f expressed in Escherichia coli.

The recently cloned cDNA for pea chloroplast thioredoxin f was used to produce, by PCR, a fragment coding for a protein lacking the transit peptide. This cDNA fragment was subcloned into a pET expression vector and used to transform E. coli cells. After induction with IPTG the transformed cells produce the protein, mainly in the soluble fraction of the broken cells. The recombinant thioredoxin f has been purified and used to raise antibodies and analysed for activity. The antibodies appear to be specific towards thioredoxin f and do not recognize other types of thioredoxin. The recombinant protein could activate two chloroplastic enzymes, namely NADP-dependent malate dehydrogenase (NADP-MDH) and fructose 1,6-bisphosphatase (FBPase), both using dithiothreitol as a chemical reductant and in a light-reconstituted/thylakoid assay. Recombinant pea thioredoxin f turned out to be an excellent catalyst for NADP-MDH activation, being the more efficient than a recombinant m-type thioredoxin of Chlamydomonas reinhardtii and the thioredoxin of E. coli. At the concentrations of thioredoxin used in the target enzyme activation assays only the recombinant thioredoxin f activated the FBPase.

Geographic distribution and evolution of yellow fever viruses based on direct sequencing of genomic cDNA fragments.

We have compared the nucleotide sequence of an envelope protein gene fragment encoding amino acids 291 to 406 of 22 yellow fever (YF) virus strains of diverse geographic and host origins isolated over a 63 year time span. The nucleotide fragment of viral RNA was examined by direct sequencing of a PCR product derived from complementary DNA. Alignment with the prototype Asibi strain sequence showed divergence of 0 to 21.5% corresponding to a maximum of 5.2% divergence in the amino acid sequence. Taking 10% nucleotide divergence as a cut-off point, the 22 YF virus strains fell into three topotypes which corresponded to different geographical areas, namely West Africa, Central-East Africa, and South America. Two subgroups were defined in West Africa, a genotypic group circulating in the sylvatic zone of the western part of Africa, from western Ivory Coast-Mali to Senegal, and a group responsible for large outbreaks from eastern Ivory Coast-Burkina Faso to Cameroon. Strains from Central-East Africa showed a low ratio of transition:transversion of about 1 instead of 8 to 10 for other strains, when their nucleotide sequences were compared with those of other African strains. This may reflect a more distant relationship between the former strains and the others. No change was observed in the highly conserved amino acid domain encompassing the TGD sequence, an important determinant of flavivirus tropism and pathogenesis. Our results support earlier observations on the genetic relationships between YF isolates established by T1 oligonucleotide fingerprinting and offer a useful tool for the understanding of YF virus distribution and evolution.