Lorette Aspart

Late Embryogenesis Abundant (LEA) protein gene regulation during Arabidopsis seed maturation

Summary This paper reviews part of our studies on gene regulation during the Arabidopsis seed maturation phase. Essentially, three complementary strategies have been used. The first one consisted in identifying genes expressed during this period by random sequencing of EST from a dry seed cDNA library and by comparing their frequency with that in an immature cDNA library. The second strategy focused on the detailed analysis of the expression of a specific group of genes coding for the class I LEA proteins, the Em genes, and analysis of their promoter. Finally we evaluated the expression of a number of LEA gene in various regulatory mutants, including abi3, lec1 and abi5 Altogether, our results illustrate the complexity of expression patterns and the interaction of various factors to define several distinct regulatory pathways.

Characterization of radish mRNA at three developmental stages

Abstract Messenger RNA was purified from immature radish seeds, dry seeds and 24-h-old seedlings. It was translated in a rabbit reticulocyte lysate in vitro protein-synthesis system. The three mRNA preparations have an overall similar activity and code for polypeptides ranging from 10 000 to 100 000 daltons. Analysis of the translation products using one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) indicates important changes in the prevalent mRNAs during seed ripening and germination. A subset of mRNA accumulates late in embryogenesis whilst most abundant immature seed mRNA decays. This late mRNA subset and a few other mRNAs detcted in immature seeds constitute the most abundant part of mRNA stored in the seed. Several less abundant mRNAs of the immature seed are also present in the dry seeds. The abundant mRNAs from dry seed disappear during early germination.

Effect of the protein synthesis inhibitor cycloheximide on RNA synthesis in radish seedlings

The effect of cycloheximide on rRNA and poly (A) (+) RNA synthesis is studied in radish seedlings. When used at a concentration of 50 microng/ml cycloheximide selectively blocks rRNA synthesis without altering poly (A) (+) RNA synthesis. Processing of the 2.3 X 10(6) dalton pre-rRNA is severely impaired. This observation should help in studies of mRNA metabolism in plants. When the concentration of cycloheximide is lowered, it is possible to completely block protein synthesis without preventing RNA synthesis. This implies that the effect of cycloheximide on RNA synthesis is probably more complex than usually assumed.

The cruciferin gene family in radish.

In order to analyse the cruciferin gene family in radish a cDNA library was screened either with heterologous rapeseed probes or by differential screening and sequencing. We could identify six partial cDNA clones belonging to two different groups of cruciferin genes which do not cross-hybridize, and probably three distinct subfamilies. One of these classes corresponds to the previously described cruciferin from rapeseed and Arabidopsis. A gene corresponding to the second group, as well as its border sequences, was isolated from a radish genomic library and analysed in more detail. The cruciferin gene (cruRS) contains three introns and encodes a 479 amino acid protein. The transcription initiation site was determined. The expression of the different group of genes was studied by northern blot analysis: genes of both classes are expressed simultaneously and roughly at the same level between 25 and 35 days after flowering. Cruciferin gene copy number was estimated by Southern blot analysis. There appear to be seven or eight genes in one class and three in the other, located at different loci.

Stability of polyadenylic and polyadenylated ribonucleic acids in radish (Raphanus sativus) seedlings

The stability of polyadenylic acid and polyadenylated RNA was investigated in young radish (Raphanus sativus) seedlings. We first studied the decay of poly(A) content, using a [3H]poly(U) assay, following a complete block of transcription by cordycepin (200 microgram/ml). Two lifetime classes of polyadenylic acid have been determined in these seedlings: a short-lived component with a half-life of 30 min which represents 60% of poly(A) and a more stable component with varying half-lives of which the majority range from 4-10 h and a few are considerably longer. During this period rRNA was shown to decay linearly, taking about 41 h for half of this RNA to disappear. The life-time of the other moiety of polyadenylated-RNA was analysed by continuous labelling with [3H]uridine. We have been able to demonstrate that a significant part of the mRNA molecules turns over with a half-life similar to that of the more slowly turning-over poly(A). No evidence could be obtained for rapidly turning-over messenger RNA. Thus the rapidly turning over poly(A) could correspond to a poly(A) turn-over independent of the remainder of the sequence. When labelling was very long, an apparent steady-state was reached and we determined the polyadenylated RNA content of seedlings to be 2.2% of whole cell RNA. Finally, these results were compared with those previously obtained in studying early germination of radish embryo axes. In contrast with stored mRNA which is rapidly degraded following imbibition, part of the mRNA present in 22 h old seedlings is stable for several hours.

STUDIES ON RIBONUCLEIC ACIDS DURING RADISH SEED GERMINATION: A SEARCH FOR POTENTIAL REGULATORY MECHANISMS

ABSTRACT Recent work on RNA metabolism during germination of radish seeds is reviewed with special emphasis on the possible regulatory mechanisms involved in the control of germination. Attention is focused on (a) the characteristics of stored mRNA and its fate during germination, (b) the time of synthesis of rRNA and polyadenylated RNA and on possible controls at the level of processing of pre-rRNA, and (c) determination of turnover of mRNA, turn-over of poly(A) and changes in turn-over associated with development.

Gene Expression During Seed Formation and Maturation in Crucifereae

Mature seeds contain a significant stock of stored mRNA, the life-span of which is as long as the seed-life (Payne, 1976; Delseny et al., 1977). A basic question in seed biology is the role and function of this stored mRNA. During the last ten years, many plant molecular biologists have addressed this question. As a result, seed development has been extensively studied. Most results concern the easiest genes to deal with, those coding for the storage proteins. However this gives only a partial view of seed development (Dure, 1985; Casey et al., 1986). Trying to answer questions concerning mRNA stored in mature seeds we have been led to analyse gene expression at various developmental stages and to realise that during seed development a number of genes are differentially expressed and sequentially switched on and off.