Summary In order to identify new factors involved in Em (a class I Late Embryogenesis Abundant protein) gene expression, Arabidopsis mutants with an altered expression of an Em promoter GUS fusion construct and a modified accumulation of Em transcripts and proteins were isolated. Germination tests on ABA showed that the most affected mutant had a weak abi phenotype. Complementation tests further revealed this mutant to be a new abi5 allele, consequently named abi5–5. In addition to reducing the final level of Em transcripts in the dry seed, the abi5–5 mutation causes a delay in the accumulation of AtEm1 during seed development. An additional characteristic of the abi5–5 mutant, is the ability of its seeds to germinate at high concentrations of salt and mannitol. The abi5–5 mutation was characterized at the molecular level and was shown to result from a two base pair deletion in the coding sequence of the ABI 5 gene. The wild type and mutant recombinant proteins were produced in E. coli and were assayed for DNA‐binding activity on their target promoters by electrophoretic mobility shift assay (EMSA). The ABI5 recombinant protein binds the ABRE sequence in the AtEm6 promoter as shown by Dnase footprinting. Among the ABRE‐type sequences selected on both Em promoters, the G‐box type AGACACGTGGCATGT element of the AtEm6 promoter shows the strongest binding by EMSA quantification.

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MATERIALS AND METHODSThe presence of auxin (2,4-D), in the culture medium of tobacco (Nicotiana tabacum var Maryland) mesophyll protoplasts is necessary both for cell wall regeneration and for passage of the cells from phase G. to phase G, of the cell cycle. Among about 250 proteins synthesized by protoplasts and characterized by their migration in a two-dimensional electrophoresis gel, 2,4-dichlorophenoxyacetic acid affects the synthesis of ll.Nine proteins are synthesized at a reduced level in the presence of the hormone, of which three are rapidly labeled and short-lived, while the others, which are long-lived, become detectable only after 2 hours of radioactive labeling, suggesting that they undergo slow posttranslational maturation. These nine proteins are proline-rich but the proline radicals are not strongly hydroxylated. The synthesis of these proteins is no longer inhibited by auxin if dichlorobenzonitril, a weed-killer which inhibits cell wall reformation of tobacco protoplasts, is added to the culture medium.Two proteins are only synthesized if protoplasts are cultivated in an auxin-containing medium. These polypeptides are rapidly labeled, and are long-lived. The inhibition of cell wall reformation by dichlorobenzonitril does not modify their synthesis.These results suggest that proteins whose synthesis is reduced by auxin are related to cell wall reformation and that they do not play a role in the induction of the cell cycle. In contrast, proteins whose synthesis is stimulated in the presence of auxin are good candidates for a role in the induction of the cell cycle.Auxin is necessary for the reformation of the cell wall as well as for the induction of mitosis. In the absence of this hormone, protoplasts initially blocked in Go phase do not progress through the other phases of the cell cycle. Using two-dimensional electrophoresis, we have previously shown (7) that this hormone partially inhibits the synthesis of several proteins and stimulates that of two others. This work, carried out using long periods of radioactive labeling (18 h), allowed the study of only stable proteins. In the present paper, we have used short labeling periods and chases to attempt to determine the synthesis pathway of these proteins and to allow the study of auxin-sensitive, shortlived proteins. In addition, we have obtained information on biochemical and physiological features of auxin-sensitive proteins. This allows us to propose hypotheses on the implication of these proteins in the phenomena of cell wall regeneration and induction of the cell cycle. obtained from New England Nuclear (Paris). Preparation of Samples. At the end of the labeling period, protoplasts were recovered by centrifugation (Janetzki TH 12; Bioblock Scientific, Strasbourg), 13,000g, 2 min, and the pellet frozen at -80°C. Soluble proteins from one pellet (one dish) were extracted for 30 min at 4°C in 100 Al 10 mm Tris-HCI (pH 7.4) containing 5 mM MgC92, 100 ug/ml pancreatic DNase, and 50 gg/ml pancreatic RNase. After centrifugation, the pellet was ree...

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When protoplasts, previously cultivated in a medium lacking auxin, are trasferd to complete medium, proteins whose synthesis is stimulated by the hormone become detectable after about 30 minutes and reach a constant level 2 to 4 hours after the beginning of hormonal treatment. In contrast, proteins whose level of synthesis is reduced by auxin, are only affected after 6 hours of treatment.Short radioactive labelings in deficient medium followed by chases in complete medium show that auxin does not interfere with posttranslational processes.Analysis of in vitro translation products of protoplast RNA shows that the time courses of auxin effects on protein synthesis and mRNA accumulation are perfectly superimposable. This allows us to exclude the possibility that auxin affects the translation process, but indicates that this hormone acts by regulating the concentration of the auxin-sensitive protein mRNAs.We have previously shown (8) that auxin induces specific modifications in the pattern ofproteins synthesized by mesophyll protoplasts during culture in vitro and have characterized the synthesis pathway of auxin-sensitive proteins (9).In this paper, we describe the time course of hormonal effects on polypeptides whose radioactivity is reduced by auxin and on polypeptides which are only present after auxin treatment. This allows us, first, to localize the auxin-dependent step in the synthesis of auxin-sensitive proteins and, second, to detect possible correlations between the hormonal effect on the protein pattern and on the physiology of the protoplasts. This has been carried out not only by transferring protoplasts from medium lacking auxin to complete medium and labeling for different times, but has also been completed by chases in order to analyze the posttranslational steps and by using in vitro synthesis directed by protoplast mRNA to look at translation and mRNA synthesis. MATERIALS AND METHODSMost experiments were performed using materials and methods described in the accompanying paper (9). We present here only additional methods. In certain experiments, proteins were separated in IEF' gels in the first dimension, rather than by NEPHGE. For this, protoplast extracts were desalted to restrict 'Abbreviations: IEF, isoelectrofocusing; NEPHGE, nonequilibrium pH gradient electrophoresis. pH shift and drift.Preparation of Samples for Electrofocusing. Protoplasts were recovered by centrifugation (1 3,000g, 2 min) and the pellet frozen at -80°C. Soluble proteins from one pellet (one dish) were extracted for 30 min at 4C in 100 ,ul 10 mm Tris-HCl (pH 7.4) containing 5 mM MgCl2, 100 ,g/ml pancreatic DNase, 50 ,g/ml pancreatic RNase. After centrifugation, the pellet was reextracted with 50 Ml of the same buffer for 15 min. The two supernatants were mixed and dialyzed in a microchamber for 4 to 16 h at 4°C against water. At the end of the desalting, NP-40 was added to 2%, ampholines (LKB 3.5-10) to 2%, fl-mercaptoethanol to 5%, and urea to saturation (9.5 M).In preliminary experiments, we tested the effect of p...

ABSTRACrMajor radish (Raphanus sativus L. cv National) proteins synthesized at the beginning of germination have been characterized by their migration in two-dimensional electrophoresis.The use of 15-minute labelings shows that these proteins are encoded by stored mRNA. phoresis patterns of polypeptides synthesized in vivo or in vitro (10), these authors have shown the presence of a specific set of mRNAs which accumulate during late embryogenesis and disappear during early germination. This work is presently the only one suggesting that some stored mRNAs code for proteins playing a specific role in early germination.We have previously detected the presence of stored mRNA in dry radish embryos and analyzed their life time (6,7). As a first step in the characterization ofproteins encoded by stored mRNA in radish, we have analyzed by two-dimensional electrophoresis the polypeptides synthesized in embryos during the first 15 min of germination and in an in vitro translation system directed by stored mRNA. These polypeptides have been compared with those synthesized during late embryogenesis and late germination and with those present in dry embryos.In this report, we show that the polypeptides synthesized during the first 15 min of germination are encoded by stored mRNA and can be classified into two sets. MATERIALS AND METHODS

Polyadenylic acid [poly (A)] is detected, characterized and quantitated in dry radish embryo axis RNA using a (3)H poly (U) probe. The amount of poly (A) gradually decreases after the onset of soaking, and, after a few hours, recovers to the initial level. This variation is shown to result from the addition of two opposed phenomena: the decay of stored poly (A) and the accumulation of newly synthesized poly (A). Stored poly (A), as well as the "in vivo" protein synthesis coded for by preformed mRNA, decreases during early germination with a half-life of two hours. As a whole, these results demonstrate that at least a fraction of the stored mRNA is translated as soon as the seed is soaked and that its role is rapidly taken over by newly-made mRNA.

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