Summary-Analysis of a synthetic ABA agonist uncovers a new family of ABA binding proteins that control signal transduction by directly regulating the activity of type 2C protein phosphatases.-PP2Cs are vital phosphatases that play important roles in abscisic acid (ABA) signaling. Using chemical genetics, we previously identified a synthetic growth inhibitor called pyrabactin. Here we show that pyrabactin is a selective ABA agonist that acts through PYR1, the founding member of a family of START proteins called PYR/PYLs, which are necessary for both pyrabactin and ABA signaling in vivo. We show that ABA binds to PYR1, which in turn binds to and inhibits PP2Cs. We therefore suggest that PYR/PYLs are ABA-receptors that function at the apex of a negative regulatory pathway that controls ABA signaling by inhibiting PP2Cs. Our results

Cell cycling plays an important role in plant development, including: (1) organ morphogenesis, (2) cell proliferation within tissues, and (3) cell differentiation. In this study we use a cyclin::beta-glucuronidase reporter construct to characterize spatial and temporal patterns of cell cycling at each of these levels during wild-type development in the model genetic organism Arabidopsis thaliana (Columbia). We show that a key morphogenetic event in leaf development, blade formation, is highly correlated with localized cell cycling at the primordium margin. However, tissue layers are established by a more diffuse distribution of cycling cells that does not directly involve the marginal zone. During leaf expansion, tissue proliferation shows a strong longitudinal gradient, with basiplastic polarity. Tissue layers differ in pattern of proliferative cell divisions: cell cycling of palisade mesophyll precursors is prolonged in comparison to that of pavement cells of the adjacent epidermal layers, and cells exit the cycle at different characteristic sizes. Cell divisions directly related to formation of stomates and of vascular tissue from their respective precursors occur throughout the period of leaf extension, so that differing tissue patterns reflect superposition of cycling related to cell differentiation on more general tissue proliferation. Our results indicate that cell cycling related to leaf morphogenesis, tissue-specific patterns of cell proliferation, and cell differentiation occurs concurrently during leaf development and suggest that unique regulatory pathways may operate at each level.

The hormone abscisic acid (ABA) modulates a variety of developmental processes and responses to environmental stress in higher plants. A collection of mutations, designated era, in Arabidopsis thaliana that confer an enhanced response to exogenous ABA includes mutations in the Era1 gene, which encodes the beta subunit of a protein farnesyl transferase. In yeast and mammalian systems, farnesyl transferases modify several signal transduction proteins for membrane localization. The era1 mutants suggest that a negative regulator of ABA sensitivity must be acted on by a farnesyl transferase to function.

SUMMARYNext-generation genomic sequencing technologies have made it possible to directly map mutations responsible for phenotypes of interest via direct sequencing. However, most mapping strategies proposed to date require some prior genetic analysis, which can be very time-consuming even in genetically tractable organisms. Here we present a de novo method for rapidly and robustly mapping the physical location of EMS mutations by sequencing a small pooled F 2 population. This method, called Next Generation Mapping (NGM), uses a chastity statistic to quantify the relative contribution of the parental mutant and mapping lines to each SNP in the pooled F 2 population. It then uses this information to objectively localize the candidate mutation based on its exclusive segregation with the mutant parental line. A user-friendly, web-based tool for performing NGM analysis is available at http://bar.utoronto.ca/NGM. We used NGM to identify three genes involved in cell-wall biology in Arabidopsis thaliana, and, in a power analysis, demonstrate success in test mappings using as few as ten F 2 lines and a single channel of Illumina Genome Analyzer data. This strategy can easily be applied to other model organisms, and we expect that it will also have utility in crops and any other eukaryote with a completed genome sequence.

z Each of these authors contributed equally to this study. SummaryGenetic screens have identi®ed a number of genes that regulate abscisic acid (ABA) responsiveness in Arabidopsis. Using a combination of suppressor screens and double mutant analysis, we have determined a genetic relationship for a number of these ABA response loci. Based on germination in the presence of exogenous ABA, the ABI1 and ABI2 phosphatases act at or upstream of the ERA1 farnesyl transferase and the ABI3 and ABI5 transcription factors act at or downstream of ERA1. In contrast with ABI3 and ABI5, the ABI4 transcription factor appears to act at or upstream of ERA1. Based on reporter gene constructs, the upstream regulation of ABI3 by ERA1 occurs at least partially at the level of transcription, suggesting that this lipid modi®cation is required to attenuate ABI3 expression. Similar experiments also indicate that ABI3 is auxin inducible in lateral root primordia. Related to this, loss-of-function abi3 alleles show reduced lateral root responsiveness in the presence of auxin and an auxin transport inhibitor, and era1 mutants have increased numbers of lateral roots. These results suggest the possibility that genes identi®ed through ABA responsive germination screens such as ERA1 and ABI3 have functions in auxin action in Arabidopsis.

A 3D atomistic model of a plant cellulose synthase (CESA) has remained elusive despite over forty years of experimental effort. Here, we report a computationally predicted 3D structure of 506 amino acids of cotton CESA within the cytosolic region. Comparison of the predicted plant CESA structure with the solved structure of a bacterial cellulose-synthesizing protein validates the overall fold of the modeled glycosyltransferase (GT) domain. The coaligned plant and bacterial GT domains share a six-stranded β-sheet, five α-helices, and conserved motifs similar to those required for catalysis in other GT-2 glycosyltransferases. Extending beyond the cross-kingdom similarities related to cellulose polymerization, the predicted structure of cotton CESA reveals that plant-specific modules (plant-conserved region and class-specific region) fold into distinct subdomains on the periphery of the catalytic region. Computational results support the importance of the plant-conserved region and/or class-specific region in CESA oligomerization to form the multimeric cellulose-synthesis complexes that are characteristic of plants. Relatively high sequence conservation between plant CESAs allowed mapping of known mutations and two previously undescribed mutations that perturb cellulose synthesis in Arabidopsis thaliana to their analogous positions in the modeled structure. Most of these mutation sites are near the predicted catalytic region, and the confluence of other mutation sites supports the existence of previously undefined functional nodes within the catalytic core of CESA. Overall, the predicted tertiary structure provides a platform for the biochemical engineering of plant CESAs.

The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1 A903V and CESA3 T942I in Arabidopsis thaliana. Using 13 C solidstate nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1 A903V and CESA3 T942I displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1 A903V and CESA3 T942I have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.cell wall | polysaccharide | quinoxyphen

Contents Summary 7 Introduction 8 Cell expansion and plant growth 8 Cell wall responses to stress 11 Pathogen attack and mechanical stimuli 11 Lessons from yeast 12 Candidate sensors and receptors in plants 14 Conclusions 17 Acknowledgements  17 References  17 Summary The emerging view of the plant cell wall is of a dynamic and responsive structure that exists as part of a continuum with the plasma membrane and cytoskeleton. This continuum must be responsive and adaptable to normal processes of growth as well as to stresses such as wounding, attack from pathogens and mechanical stimuli. Cell expansion involving wall loosening, deposition of new materials, and subsequent rigidification must be tightly regulated to allow the maintenance of cell wall integrity and co‐ordination of development. Similarly, sensing and feedback are necessary for the plant to respond to mechanical stress or pathogen attack. Currently, understanding of the sensing and feedback mechanisms utilized by plants to regulate these processes is limited, although we can learn from yeast, where the signalling pathways have been more clearly defined. Plant cell walls possess a unique and complicated structure, but it is the protein components of the wall that are likely to play a crucial role at the forefront of perception, and these are likely to include a variety of sensor and receptor systems. Recent plant research has yielded a number of interesting candidates for cell wall sensors and receptors, and we are beginning to understand the role that they may play in this crucial aspect of plant biology.