The hydra mutants of Arabidopsis are characterized by a pleiotropic phenotype that shows defective embryonic and seedling cell patterning, morphogenesis, and root growth. We demonstrate that the HYDRA1 gene encodes a ⌬ 8-⌬ 7 sterol isomerase, whereas HYDRA2 encodes a sterol C14 reductase, previously identified as the FACKEL gene product. Seedlings mutant for each gene are similarly defective in the concentrations of the three major Arabidopsis sterols. Promoter::reporter gene analysis showed misexpression of the auxin-regulated DR5 and ACS1 promoters and of the epidermal cell file-specific GL2 promoter in the mutants. The mutants exhibit enhanced responses to auxin. The phenotypes can be rescued partially by inhibition of auxin and ethylene signaling but not by exogenous sterols or brassinosteroids. We propose a model in which correct sterol profiles are required for regulated auxin and ethylene signaling through effects on membrane function. INTRODUCTIONSterols are essential components of fungal, plant, and animal membranes. They regulate fluidity and interact with lipids and proteins within the membrane, and they are the precursors for the brassinosteroid (BR) hormones in plants (Hartmann, 1998). The sterol biosynthetic pathway in plants, therefore, can be viewed as comprising two parts: one branch produces the bulk membrane sterols (the principal sterols in Arabidopsis being stigmasterol, campesterol, and sitosterol), and the second part represents the BR synthesis branch. Sterol biosynthesis has been well characterized in yeast, supported by a powerful system of genetic analysis. In animals, and more recently in plants, sterol biosynthetic enzyme function has been confirmed via the functional complementation of yeast mutants (Gachotte et al., 1996). Functional analysis of sterol function in plants has involved a range of approaches, but recently, genetic studies have provided useful information on the requirement for particular enzymes in sterol and BR biosynthesis and, for BRs, perception and signal transduction (Clouse, 2000;Diener et al., 2000;Schaeffer et al., 2001).In animals, sterols appear to be important to maintain correct cell-signaling activities. For example, drugs such as the ligand SR31747A, which inhibits the activity of the receptor (emopamil binding protein [EBP], which has ⌬ 8-⌬ 7 sterol isomerase activity), cause defects in a diversity of cellular processes, including the inhibition of mammalian lymphocyte proliferation in response to mitogens (Derocq et al., 1995) and the inhibition of graft rejection in mouse via the modulation of gene expression (Carayon et al., 1995), and they may influence lipoprotein functions leading to immunosuppressive effects (Dussossoy et al., 1999). In plants, a lack of detailed pharmacological studies has precluded analogous investigations of the role of sterols in plant cell biology.However, mutational and transgenic studies have given new insight into the roles of sterols in plant development. sterol methyltransferase1 ( smt1 ) mutants accumulate cholesterol...

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BackgroundThe control of vascular tissue development in plants is influenced by diverse hormonal signals, but their interactions during this process are not well understood. Wild-type sterol profiles are essential for growth, tissue patterning and signalling processes in plant development, and are required for regulated vascular patterning.Methodology/Principal FindingsHere we investigate the roles of sterols in vascular tissue development, through an analysis of the Arabidopsis mutants hydra1 and fackel/hydra2, which are defective in the enzymes sterol isomerase and sterol C-14 reductase respectively. We show that defective vascular patterning in the shoot is associated with ectopic cell divisions. Expression of the auxin-regulated AtHB8 homeobox gene is disrupted in mutant embryos and seedlings, associated with variably incomplete vascular strand formation and duplication of the longitudinal axis. Misexpression of the auxin reporter proIAA2∶GUS and mislocalization of PIN proteins occurs in the mutants. Introduction of the ethylene-insensitive ein2 mutation partially rescues defective cell division, localization of PIN proteins, and vascular strand development.ConclusionsThe results support a model in which sterols are required for correct auxin and ethylene crosstalk to regulate PIN localization, auxin distribution and AtHB8 expression, necessary for correct vascular development.

The roles of sterols in plant development are not well understood, but evidence is emerging that they are required for cell division, polarity and patterning by mechanisms that are independent of brassinosteroids, of which they are precursors. Previous evidence shows that two sterol-defective mutants of Arabidopsis thaliana (L.) Heynh., hyd1 and fk(hyd2), are defective in root development. Here we show that the HYD1 gene, like the FK gene, is transcriptionally active in both primary and lateral root meristems, though not in the shoot apical meristem. The patterns of cell division during early stages of lateral root initiation in the hyd1 and fk(hyd2) mutants appear normal. Previous evidence also suggests that auxin and ethylene signalling is defective in the mutants. Here we show that the cytokinin- and ethylene-responsive ACS1::GUS reporter in the fk(hyd2) mutant responds to exogenous cytokinins but not to the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, indicative of normal cytokinin signalling but supporting the hypothesis that ethylene signalling is defective. The defective root meristem cell division activity and expression patterns of the auxin-regulated DR5::GUS and IAA2::GUS reporters can be rescued to a significant extent by the pharmacological or genetic inhibition of ethylene signalling, but not by treatment with aminoethoxyvinylglycine, an inhibitor of ethylene synthesis. This supports the emerging view that the hyd1 and fk(hyd2) mutants exhibit an enhanced and unregulated ethylene signalling activity, which accounts for at least part of the observed mutant phenotypes, including disrupted auxin signalling. The possible relationship between ethylene signalling, membrane sterols and meristem function is discussed.

Summary• The AGC protein kinase OXI1 is a key protein in plant responses to oxidative signals, and is important for two oxidative burst-mediated processes: basal resistance to microbial pathogens and root hair growth. To identify possible components of the OXI1 signalling pathway, phosphoproteomic techniques were used to detect alterations in the abundance of phosphorylated proteins and peptides in an oxi1 null mutant of Arabidopsis thaliana.• The relative abundance of phosphorylated proteins was assessed either using two-dimensional gel electrophoresis and staining with the phosphoprotein stain Pro-Q Diamond or by the identification and quantification, by mass spectrometry, of stable-isotope labelled phosphopeptides.• A number of proteins show altered phosphorylation in the oxi1 mutant. Five proteins, including a putative F-box and 3-phosphoinositide-dependent kinase 1, show reduced phosphorylation in the oxi1 mutant, and may be direct or indirect targets of OXI1. Four proteins, including ethylene insensitive 2 and phospholipase D-gamma, show increased phosphorylation in the oxi1 mutant.• This study has identified a range of candidate proteins from the OXI1 signalling pathway. The diverse activities of these proteins, including protein degradation and hormone signalling, may suggest crosstalk between OXI1 and other signal transduction cascades.