Brassinosteroids (BRs) are biosynthesized from campesterol via several cytochrome P450 (P450)-catalyzed oxidative reactions. We report the functional characterization of two BR-biosynthetic P450s from Arabidopsis thaliana: CYP90C1/ ROTUNDIFOLIA3 and CYP90D1. The cyp90c1 cyp90d1 double mutant exhibits the characteristic BR-deficient dwarf phenotype, although the individual mutants do not display this phenotype. These data suggest redundant roles for these P450s. In vitro biochemical assays using insect cell-expressed proteins revealed that both CYP90C1 and CYP90D1 catalyze C-23 hydroxylation of various 22-hydroxylated BRs with markedly different catalytic efficiencies. Both enzymes preferentially convert 3-epi-6-deoxocathasterone, (22S,24R)-22-hydroxy-5a-ergostan-3-one, and (22S,24R)-22-hydroxyergost-4-en-3-one to 23-hydroxylated products, whereas they are less active on 6-deoxocathasterone. Likewise, cyp90c1 cyp90d1 plants were deficient in 23-hydroxylated BRs, and in feeding experiments using exogenously supplied intermediates, only 23-hydroxylated BRs rescued the growth deficiency of the cyp90c1 cyp90d1 mutant. Thus, CYP90C1 and CYP90D1 are redundant BR C-23 hydroxylases. Moreover, their preferential substrates are present in the endogenous Arabidopsis BR pool. Based on these results, we propose C-23 hydroxylation shortcuts that bypass campestanol, 6-deoxocathasterone, and 6-deoxoteasterone and lead directly from (22S,24R)-22-hydroxy-5a-ergostan-3-one and 3-epi-6-deoxocathasterone to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol.

Endomembrane trafficking relies on the coordination of a highly complex, dynamic network of intracellular vesicles. Understanding the network will require a dissection of cargo and vesicle dynamics at the cellular level in vivo. This is also a key to establishing a link between vesicular networks and their functional roles in development. We used a high-content intracellular screen to discover small molecules targeting endomembrane trafficking in vivo in a complex eukaryote, Arabidopsis thaliana. Tens of thousands of molecules were prescreened and a selected subset was interrogated against a panel of plasma membrane (PM) and other endomembrane compartment markers to identify molecules that altered vesicle trafficking. The extensive image dataset was transformed by a flexible algorithm into a marker-by-phenotype-by-treatment time matrix and revealed groups of molecules that induced similar subcellular fingerprints (clusters). This matrix provides a platform for a systems view of trafficking. Molecules from distinct clusters presented avenues and enabled an entry point to dissect recycling at the PM, vacuolar sorting, and cell-plate maturation. Bioactivity in human cells indicated the value of the approach to identifying small molecules that are active in diverse organisms for biology and drug discovery.chemical genomics | high content screen | endosidin | endosome T he coordination of multicellular growth to establish and maintain the morphology of eukaryotic organisms during development is orchestrated by complex regulatory processes in which vesicular trafficking within the endomembrane system is essential (1, 2). The endomembrane system is a network of interconnected pathways required to establish signaling, cell-tocell communication, cell polarity, and cell shape in response to developmental or environmental stimuli (3). Eukaryotic cells possess the ability to internalize their plasma membrane (PM) and thus rapidly remodel their protein content (4), which is essential for polar growth, cytokinesis, hormone perception and transport, response to pathogens, and metal detoxification (5-8).To dissect the endomembrane network from a systems perspective and to understand protein functions within the network, it is necessary to perturb trafficking in a controlled fashion and to examine the consequences on growth and development. The ability to induce and evaluate subcellular phenotypes at a significant scale based on combinations of multiple endomembranespecific markers is critical for characterization of the network. In this regard, small molecules are promising for the efficient discovery and evaluation of complex intracellular phenotypes because, for any molecule, lines expressing different endomembrane fluorescent protein markers can be efficiently interrogated in parallel (3).In a previous pilot screen we identified endosidin 1 (ES1), a compound that arrests PIN2 and BRI1 in SYP61 aggregates, named ES1 bodies (9). We have used a modified high-throughput confocal-based screen focused on the rapid recycling of PM ...