SummaryGenetic approaches using Arabidopsis thaliana aimed at the identification of mutations affecting events involved in auxin signalling have usually led to the isolation of auxinresistant mutants. From a selection screen specifically developed to isolate auxin-hypersensitive mutants, one mutant line was selected for its increased sensitivity to auxin (⍥ 2 to 3) for the root elongation response. The genetic analysis of sax1 (hypersensitive to abscisic acid and auxin) indicated that the mutant phenotype segregates as a single recessive Mendelian locus, mapping to the lower arm of chromosome 1. Sax1 seedlings grown in vitro showed a short curled primary root and small, round, dark-green cotyledons. In the greenhouse, adult sax1 plants were characterized by a dwarf phenotype, delayed development and reduced fertility. Further physiological characterization of sax1 seedlings revealed that the most striking trait was a large increase (ϫ 40) in ABA-sensitivity of root elongation and, to a lesser extent, of ABA-induced stomatal closure; in other respects, hypocotyl elongation was resistant to gibberellins and ethylene. These alterations in hormone sensitivity in sax1 plants co-segregated with the dwarf phenotype suggesting that processes involved in cell elongation are modified. Treatment of mutant seedlings with an exogenous brassinosteroid partially rescued a wild-type size, suggesting that brassinosteroid biosynthesis might be affected in sax1 plants. Wild-type sensitivities to ABA, auxin and gibberellins were also restored in sax1 plants by exogenous

Abstract. Elicitor-induced cytoplasmic pH changes of tobacco (Nicotiana tabacum L. cv. Xanthi) cells grown in suspension cultures were explored under a variety of conditions by using a flexible technique based on the distribution of [14C] benzoic acid between the intracellular and extracellular compartments. Comparison of data obtained by this technique and by 31p-nuclear magnetic resonance spectrometry qualifies the benzoic acid distribution method as a convenient and reliable way to probe cytoplasmic pH variations. Various elicitors shown to induce several defense-related responses in tobacco cells, namely oligogalacturonides of degree of polymerization 7-20, pectolyase from Aspergillusjaponicus, Phytophthora megasperma crude elicitors and purified cryptogein, triggered cytoplasmic acidifications differing in intensity and kinetics according to the signal molecule. In contrast, no changes in cytoplasmic protons and external pH were observed in cells treated with short galacturonide oligomers, or with soybean-specific hepta 13-glucoside from P. megasperma, which are devoid of elicitor activity in tobacco cells. The oligogalacturonide-induced cytoplasmic acidification was inhibited by two structurally unrelated protein kinase inhibitors, staurosporine and 6-dimethylaminopurine, which both reduced the external alkalinization response to the elicitor. The protein phosphatase inhibitor calyculin A alone behaved as an elicitormimicking molecule in triggering cytoplasmic acidification, again associated with extracellular alkalinization. These results indicate that the increase in the cytoplasmic concentration of protons may be considered as a common early intracellular response of tobacco cells to elicitors, associated with the extracellular alkalinization response and controlled by protein phosphorylation.Abbreviations: BA(H) = benzoic acid (protonated form); 6-DMAP = 6-dimethylaminopurine; DP = degree of polymerization; Mes = 2-(N-morpholino)ethanesulfonic acid; OG = oligogalacturonide; pHc = cytoplasmic pH; 31p-NMR = nuclear magnetic resonance spectroscopy of 31p atoms Correspondence to: Y. Mathieu; FAX: 33 (1) 69 82 37 68

SummaryIn this issue we described a dwarf mutant in Arabidopsis thaliana, sax1, which is affected in brassinosteroid biosynthesis. This primary defect is responsible for alterations in hormone sensitivity of sax1 plants characterized by the hypersensitivity of root elongation to abscisic acid and auxin and the insensitivity of hypocotyl growth to gibberellins and ethylene (Ephritikhine et al., 1999; Plant J. 18, 303-314). In this paper, we report the further characterization of the sax1 mutant aimed at identification of the mutated step in the brassinosteroid biosynthesis pathway. Rescue experiments with various intermediates of the pathway showed that the sax1 mutation alters a very early step catalyzing the oxidation and isomerization of 3β-hydroxyl,∆ 5,6 precursors to 3-oxo,∆ 4,5 steroids. The mapping of the mutation, the physiological properties of the mutant and the rescue experiments indicate that sax1 defines a new locus in the brassinosteroid biosynthesis pathway. The SAX1 protein is involved in brassinosteroiddependent growth of seedlings in both light and dark conditions.

When auxin was omitted during either the preparation or the culture of tobacco mesophyll protoplasts, as well as during both periods, synthesis of ,B-glucanase was spontaneously induced. In contrast, when protoplasts were prepared and cultured in the presence of 16 micromolar 1-naphthaleneacetic acid (optimal concentration for protoplast division), the expression of ,B-glucanase was maintained close to the minimal level observed in tobacco leaves. This inhibitory effect was only promoted by active auxins (1 -naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and 3-indoleacetic acid) but not by inactive auxin analogs. Tobacco protoplasts responded to exogenous elicitors from the cell wall of Phytophthora megasperma glycinea (Pmg) by accumulating 0-glucanase in the presence of 16 micromolar 1-naphthaleneacetic acid. At higher auxin concentrations, the elicitor-induced ,B-glucanase synthesis was inhibited. Naphthaleneacetic acid concentration (3 x 10-5 molar) required to inhibit by 50% the expression of this defense reaction triggered by a near-optimal elicitor concentration was about 100 times higher than that sufficient to inhibit by 50% the spontaneous expression in nonelicited protoplasts. This is the first demonstration of an auxin-fungal elicitor interaction in the control of a defined defense reaction. The above observations were extended to soybean cell protoplasts. The Pmg elicitor-induced stimulation of the synthesis of pathogenesis related P17 polypeptides and of a 39-kilodalton peptide immunologically related to tobacco ,Bglucanase was only observed when the spontaneous accumulation of these proteins was inhibited in auxin-treated protoplasts.

Cytoplasmic acidi®cation has been previously characterized as a common intracellular response of tobacco cells to elicitors (Y. Mathieu et al. 1996, Planta 199: 416±424). The possibility that cytosolic protons may activate plant defense reactions through the increased mRNA transcript levels of speci®c genes was investigated in suspension-cultured tobacco (Nicotiana tabacum L. cv. Xanthi) cells. Transcript levels of phenylalanine ammonia-lyase and 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the ®rst enzymes of phenylpropanoid and tobacco phytoalexin pathways, respectively, were quanti®ed in a variety of conditions leading to intracellular acidi®cation. Three arti®cial ways of inducing intracellular acidi®cation, acid loading, inorganic phosphate uptake and inhibition of the plasma-membrane ATPase were able to increase transcript levels of the two genes in certain conditions. The combination of two parameters of the intracellular pH decrease, intensity and duration, was shown to be crucial for the induction of the two kinds of transcript. In particular, experiments using addition of two signals showed that a cytosolic acidi®cation that was too long and/or too intense no longer allowed the increase in mRNA transcripts to occur. Together, the results indicate that cytosolic protons take part in the accumulation of the two defense gene transcripts and may be considered as one of the second messengers involved in the transduction of elicitor molecules in tobacco cells.

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Children with Alagille syndrome show high serum cholesterol (15-20 mmol/L). To establish correlation of this unusual level of cholesterol with the regulation of cholesterol metabolism, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity and synthesis of cholesterol, fatty acids and acidic steroids from [14C]acetate were determined in cultured skin fibroblasts from 2-3 year old children. Prostaglandin E2 (PGE2) synthesis and nucleic acid synthesis were determined in cells when they were growing in medium containing normal, Alagille or fetal bovine serum. These values were similar to values of controls. HMGR activity was found to be similar in cells of control and children with the syndrome, whether the cells were incubated in lipoprotein-deficient or normal medium. Incorporation of acetate into cholesterol was inhibited to a greater extent by lipoprotein-containing medium in control than in children with the syndrome. Fatty acid synthesis was similar in all conditions. 1-7% of the recovered lipid radioactivity in cells and medium separated as acidic steroids. Serum from a donor patient, when included in the medium, did not affect PGE2 or nucleic acid synthesis compared with normal human or fetal bovine serum. The data suggest that cells of children with Alagille syndrome may have a membrane defect of transfer of cholesterol (LDL receptor defect) leading to excessive cholesterol synthesis. Also, synthesis of acidic steroids (bile acid-like material) and their secretion into the medium occurs in normal fibroblasts and those from children with the syndrome.

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