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.

AKRP and EMB506 are two ankyrin repeat proteins essential for plastid differentiation and plant development in Arabidopsis.

EMB506 is a chloroplast protein essential for embryo development, the function of which is unknown. A two-hybrid interaction screen was performed to provide insight into the role of EMB506. A single interacting partner, AKRP, was identified among a cDNA library from immature siliques. The AKR gene (Zhang et al., 1992, Plant Cell 4, 1575-1588) encodes a protein containing five ankyrin repeats, very similar to EMB506. Protein truncation series demonstrated that both proteins interact through their ankyrin domains. Using reverse genetics, we showed that loss of akr function resulted in an embryo-defective (emb) phenotype indistinguishable from the emb506 phenotype. Transient expression of the signal peptide of AKRP fused to green fluorescent protein demonstrated the chloroplast localization of AKRP. The ABI3 promoter was used to express AKR in a seed-specific manner in order to analyse the post-embryonic effect of AKR loss of function in akr/akr seedlings. Homozygous fertile and viable akr/akr plants were obtained. These plants exhibited mild to severe defects in chloroplast and leaf cellular organization. We conclude that EMB506 and AKRP are involved in crucial and tightly controlled events in plastid differentiation linked to cell differentiation, morphogenesis and organogenesis during the plant life cycle.

Partial complementation of embryo defective mutations: a general strategy to elucidate gene function.

The EMB 506 gene has been characterised as essential for embryo development. To provide insights into the role of EMB 506, which is hidden by the embryo defective phenotype, the ABI3 promoter was fused to the EMB 506 cDNA. The expression of such a transgene should provide sufficient protein during embryogenesis to ensure normal embryo development in homozygous emb 506 seeds. We show that homozygous emb 506 seedlings, partially complemented with the ABI3::EMB 506 transgene, can be obtained. Most of the rescued emb 506 plants are able to flower and to set normal seeds, but show mild to severe depigmentation of rosette leaves and/or inflorescences. This effect on chloroplast development indicated a putative chloroplast localisation of the EMB 506 protein, which was demonstrated by GFP-protein fusion. However, EMB 506 cannot be considered as a chloroplast housekeeping protein only, since EMB 506 is not present in all photosynthetic tissues. This study demonstrates the power of this simple strategy, which could be widely applied to other emb mutants and which may reveal similar or additional roles for EMB genes at vegetative stages of the life cycle.

Differential expression of the Arabidopsis genes coding for Em-like proteins.

Late embryogenesis abundant (lea) genes are a large and diverse group of genes highly expressed during late stages of seed development. Five major groups of LEA proteins have been described. Two Em genes (group I lea genes) are present in the genome of Arabidopsis thaliana L., AtEm1 and AtEm6. Both genes encode for very similar proteins which differ basically in the number of repetitions of a highly hydrophilic amino acid motif. The spatial patterns of expression of the two Arabidopsis Em genes have been studied using in situ hybridization and transgenic plants transformed with the promoters of the genes fused to the beta-glucuronidase reporter gene (uidA). In the embryo, AtEm1 is preferentially expressed in the pro-vascular tissues and in meristems. In contrast, AtEm6 is expressed throughout the embryo. The activity of both promoters disappears rapidly after germination, but is ABA-inducible in roots of young seedlings, although in different cells: the AtEm1 promoter is active in the internal tissues (vasculature and pericycle) whereas the AtEm6 promoter is active in the external tissues (cortex, epidermis and root hairs). The AtEm1 promoter, but not AtEm6, is also active in mature pollen grains and collapsed nectaries of young siliques. These data indicate that the two Em proteins could carry out at least slightly different functions and that the expression of AtEm1 and AtEm6 is controlled at, at least, three different levels: temporal, spatial and hormonal (ABA).

The BANYULS gene encodes a DFR-like protein and is a marker of early seed coat development.

Mutations in the BANYULS (BAN) gene lead to precocious accumulation of anthocyanins in immature seed coat in Arabidopsis. The ban -1 allele has been isolated from a collection of T-DNA transformants and found to be tagged by the integrative molecule. The sequencing of wild-type and two independent mutant alleles confirmed the identity of the gene. Analysis of the full-length cDNA sequence revealed an open reading frame encoding a 342 amino acid protein which shared strong similarities with DFR and other enzymes of the phenylpropanoid biosynthesis pathway. BAN expression was restricted to the endothelium of immature seeds at the pre-globular to early globular stages of development as predicted from the maternal inheritance of the phenotype, and therefore represents a marker for early differentiation and development of the seed coat. BAN is probably involved in a metabolic channelling between the production of anthocyanins and pro-anthocyanidins in the seed coat.

The EMB 506 gene encodes a novel ankyrin repeat containing protein that is essential for the normal development of Arabidopsis embryos.

The EMB 506 gene of Arabidopsis, required for the normal development of the embryo beyond the globular stage, has been cloned. The gene encodes a protein of predicted size 35 kDa that contains five ankyrin (ANK) repeats within the C terminal moiety. ANK repeats are conserved domains of 33 amino acids involved in specific recognition of protein partners. The EMB 506 protein was detected at different stages of silique development but accumulated preferentially in the mature cauline leaves. The rescue of homozygous emb 506 embryos by complementation with the wild-type sequence cDNA demonstrated that the emb mutation is a consequence of the T-DNA insertion and that integration and expression of the transgene occurred during gametogenesis and/or early embryo development. In addition to the drastic effect of the emb 506 mutation during embryo development, complementation experiments revealed another effect of the gene: emb 506 plants transformed with the wild-type EMB 506 sequence were able to produce viable seeds but showed a reduction of apical dominance and the presence of adventitious buds or bracts along the stem. This result supports the idea that genes essential for embryogenesis may also be required at other stages of the plant life cycle.

BANYULS, a novel negative regulator of flavonoid biosynthesis in the Arabidopsis seed coat.

A mutant of Arabidopsis that accumulates a high level of red pigments within the seed coat has been isolated from a population of T-DNA-transformed plants. Genetic analysis revealed that the mutation is recessive and affects maternal seed tissues only. Due to the color of the immature seeds, this mutation was named banyuls (ban). Pigments accumulated continuously from early seed development to the desiccation stage in the seed coat of the mutant. The phenotype of the double mutant banyuls/ transparent testa confirmed the flavonoid nature of the pigments and enabled assignment of the regulatory TT (Transparent Testa) genes to two groups according to their epistatic relationship to ban. The flavonoid content of germinated ban and wild-type seedlings was similar. Plants harbouring the ban mutation had a normal formation of trichomes and root hairs and were not affected in their responses to light. The seeds of ban plants exhibited reduced germination compared to wild-type which may be a direct consequence of the high level of pigments. These results suggest that BANYULS functions as a negative regulator of flavonoid biosynthesis that prevents accumulation of pigments in the seed coat during early embryogenesis in Arabidopsis.

Induction and expression of seed-specific promoters in Arabidopsis embryo-defective mutants.

In order to assess the importance of morphogenesis on the induction of promoter markers for storage and Lea programmes, advantage was taken of the emb mutations producing embryos arrested at a wide range of developmental stages in Arabidopsis. These embryos are viable during their stage of developmental arrest and continue to divide further, but apparently without further differentiation into the main organs and tissues of the normal embryos. Eight independent emb mutants arrested in their development prior to the cotyledon stage were selected. These emb embryos lack the normal morphology of the wild-type embryos when the synthesis of storage and Lea proteins are normally initiated. The 2S1-uidA chimeric gene, representative of the maturation programme and the Em 1-uidA chimeric gene, representative of the desiccation programme were introduced by crosses into the emb background. In the eight emb lines, the expression of the GUS reporter gene directed by the 2S1 and Em 1 promoters was observed in the aborted seeds irrespective of their stage of developmental arrest. The time of induction of the expression of both promoters was the same in the arrested embryos as compared with the normal embryos within the same silique. Thus, the activation of these two promoters is triggered by the same signal and can occur in the absence of morphogenesis. However, in the absence of normal organ formation, the expression of the reporter gene under the control of the 2S1 and Em1 promoters was evident throughout the whole seed tissues. In normal seed development, the hormone abscisic acid (ABA) activates the promoters of the 2S1 and Em 1 genes. One of the important members of the signal transduction pathway of ABA is the ABI3 protein. It has been shown previously that this protein is a prerequisite for the induction of Em 1 by ABA in seeds. A good correlation with the expression of the ABI3 promoter and the 2S1 and Em 1 promoters was found in emb seeds tissues. This observation suggests that the promoters of the 2S1 and the Em 1 genes are expressed in the mutant seeds not at a basal level, but are probably induced by ABA, as in normal seed development.

Serum selenium level in patients with colorectal cancer.

Serum selenium levels were determined by fluorometric procedure in 37 patients of both sexes suffering from colorectal cancer. The diagnosis was verified with histopathological examination during surgical treatment. The values found were 46.8 +/- 11.2 micrograms/L. The control group consisted of 230 healthy persons from the same environment as the group of patients. The values found were 64.2 +/- 11.5 micrograms/L. The results of this study are compared with the results of the other research groups analyzing the level of selenium in colorectal cancer.

Cucumber mosaic virus satellite RNA (Y strain): analysis of sequences which affect yellow mosaic symptoms on tobacco.

Plants infected with cucumber mosaic virus (CMV) (KIN strain) produce a mild mosaic disease on tobacco whereas infections of CMV with satellite RNA (strain Y) cause a severe yellow mosaic. Analysis of recombinant and mutant forms of satellite RNA identified a site (nucleotides 185/186) in the Y satellite RNA that affects the ability to induce the yellow mosaic in combination with CMV but not with tomato aspermy virus. The location of this site with respect to other mutations in the satellite RNA indicated that polypeptides, which may be encoded by the satellite RNA, have no role in induction of yellow mosaic symptoms. The symptom induction is therefore an effect of the satellite RNA on the host plant with the intervention of the helper virus. In the course of the mutation analysis of satellite RNA we detected several secondary mutations which arose in planta. Two of these were deletions of more than 80 nucleotides. Other forms of mutant satellite RNA were non-functional even though the modifications involved nucleotides completely within the large secondary deletions. These data imply complex intramolecular interactions in the satellite RNA.

Cucumber mosaic virus satellite RNA (strain Y): analysis of sequences which affect systemic necrosis on tomato.

The location of a sequence within the Y satellite RNA of cucumber mosaic virus (CMV) that confers the ability to induce necrosis on tomato plants has been analysed using chimeric satellite RNAs. These recombinant RNA molecules contained parts of the Y (necrogenic) and Ra (benign) satellite RNAs and were inoculated into tomato plants together with CMV helper virus. From the composition of the recombinant satellite RNAs that induced necrosis it was concluded that, of the nucleotides which differ between Y and Ra satellite RNAs, those affecting necrosis are on the 3' side of nucleotide 259. The composition of satellite RNAs that failed to induce necrosis implies that at least some of the necrogenic positions are on the 3' side of nucleotide 311. The symptoms induced by mutated forms of Y and Ra satellite RNAs showed that nucleotide spacing between positions 322 and 323 and sequence identity at one or more of nucleotides 318, 323 or 325 affects the necrogenic potential of Y satellite RNA. The effect of a frameshifting mutation in Y satellite RNA and the location of the necrogenic sites relative to open reading frames in other satellite RNAs suggested that necrosis is not caused by polypeptides encoded in satellite RNA.

Serum selenium levels in untreated children with acute lymphoblastic leukemia. I.

Serum selenium in the control group of 79 children aging 1 to 16 years was measured. A slight increase in serum selenium values with age occurred. The 89 patients of the test group were suffering from malignant proliferative diseases. The largest number of patients was suffering from acute lymphatic leukemia (78 patients), then Hodgkin's and non-Hodgkin's lymphoma (5 patients) and other malignant proliferative diseases (6 patients). Samples were taken before any therapeutic treatment at the time of diagnosis. Patients were of the same ages as the control group. Statistical analysis of the data shows that a significant difference exists in the selenium level of most of the patients as compared with the control group. They have a lower serum selenium. Only patients with Hodgkin's and non-Hodgkin's lymphoma have same selenium content as the control group.

Symptom production on tobacco and tomato is determined by two distinct domains of the satellite RNA of cucumber mosaic virus (strain Y).

Complementary DNAs of two different satellite RNA isolates from cucumber mosaic virus (CMV), Y from Japan and Ra from France, have been cloned in a transcription vector containing the Pr promoter. When inoculated on plants with CMV RNA (strain KIN), the transcripts of the cloned Y satellite cDNA elicit a bright yellow mosaic on tobacco and a lethal necrosis on tomato. Addition of the transcripts of the Ra satellite cDNA to an inoculum of CMV RNA resulted in symptom attenuation on both tobacco and tomato, in agreement with the characterized symptoms of the natural satellite. Recombinant molecules involving these two satellites have been constructed in order to determine which parts of the Y satellite RNA are involved in symptom induction. The determinant for symptom production on tobacco lies in the region between nucleotides 1 and 219. The domain for necrotic symptoms on tomato resides on the 3' half of the molecule beyond nucleotide 219.

[HLA and narcolepsy. Apropos of 28 cases including 2 negative HLA-DR2]. / HLA et narcolepsie. A propos de 28 cas dont deux HLA-DR2 négatifs.

Association between narcolepsy and HLA-DR2 antigen is the strongest so far described between an HLA antigen and a disease. Among 28 narcoleptic patients, we found two HLA-DR2 negative cases: a caucasoid woman also suffering from dystrophia myotonica and a negroid. All of our patients were HLA-DQW1 positive. An hypothetical narcolepsy susceptibility gene could be located in the HLA region, closer to the DQ than to the DR gene. It could be a pathologic allele of a sleep controlling gene in linkage disequilibrium with DQW1. Presence of DQW1 is a quasi-requisite for the expression of narcolepsy. It is not sufficient as it is observed in 70 p. 100 of controls.

[South-Eastern France, a high risk area for multiple sclerosis?]. / Le Sud-Est français, zone "à haut risque" de sclérose en plaques?

A questionnaire-based prevalence study was conducted in the Chalon-sur-Saône and Avignon areas, in the Rhône-Saône valley, France, to determine the frequency of multiple sclerosis. These areas are 300 km apart and lie on the 47 degrees and 44 degrees North parallels respectively. Age-adjusted prevalence rates on March 20, 1984 were 58.5 and 48.6 per 100,000 inhabitants respectively. There was no significant difference between the two areas. These preliminary data suggest that south-eastern France, as represented by Avignon, may fall within the high risk area for multiple sclerosis.

[Dexamethasone suppression test in psychiatric pathology]. / Test de freinage à la dexaméthasone en pathologie psychiatrique.

The dexamethasone suppression test (DST) has been used in psychiatric pathology for about 10 years. Carroll et al. consider this test to be specific of endogenous depression. According to these, and many other authors, approximately 55% of patients with endogenous depression show a positive response to the test, whereas a positive response is observed in only 4% of normal subjects or patients with psychiatric disorders other than major depressive disorders. The DST was performed in 162 psychiatric inpatients (5 with organic disease, 28 with schizophrenic disorders, 17 with major affective disorders, 5 with obsessive compulsive disorders, 103 with dysthymic disorders and 4 unclassified). Dexamethasone (1 mg) was administered orally at 11 p.m., and plasma cortisol concentration was measured the following day at 16 p.m. Response to the test was positive in 53% of patients with major affective disorders, 25% of those with schizophrenic disorders, 60% with obsessive compulsive disorders and 18% with dysthymic disorders. There was no statistical difference in the results according to age, sex ratio, family history of depression or duration of the disorders. Only two variables were close to the P less than 0.05 level of statistical significance: severity of the disorders and early morning awakening. DST sensitivity, therefore, would appear to be about 50% in major affective disorders, but this test is not specific as it may also be positive in other psychiatric disorders. A positive dexamethasone suppression test may be regarded as a sign of severity of psychiatric disorders.

Plasma cells in cerebrospinal fluid and multiple sclerosis: diagnostic yield and clinicobiological correlations.

Cerebrospinal fluid (CSF) cytocentrifugation was performed for plasma cells' demonstration in parallel with white cell count (WCC) and quantitative protein assays. Over a 5-year period, 154 consecutive multiple sclerosis (MS) patients were studied and compared to 28 other inflammatory neurological disease, 85 non-inflammatory neurological disease and 29 non-neurological disease cases. CSF cytology was easy to perform, gave definitive results within 2 h and was abnormal in 80 MS patients, 26 of whom had a normal WCC. Its sensitivity in MS was 0.57, i.e. higher than for WCC (0.45) but lower than for IgG index (0.70) and IgG synthesis rate (0.71). Its specificity was 0.86, not significantly different from specificity of WCC, IgG index and IgG synthesis rate. Plasma cells demonstration in MS CSF was neither a disease activity nor a prognosis marker. It was significantly correlated with pleiocytosis and intrathecal IgG synthesis. If these morphologically defined plasma cells are actual B cells, they could represent circulating individuals of the lymphocyte clones active in MS plaques and have a pathogenetic significance.