Tandem C2 domains mediate dynamic organelle targeting of a DOCK family guanine nucleotide exchange factor.

Multicellular organisms use dedicator of cytokinesis (DOCK) family guanine nucleotide exchange factors (GEFs) to activate Rac/Rho-of-plants small GTPases and coordinate cell shape change. In developing tissues, DOCK signals integrate cell-cell interactions with cytoskeleton remodeling, and the GEFs cluster reversibly at specific organelle surfaces to orchestrate cytoskeletal reorganization. The domain organizations among DOCK orthologs are diverse, and the mechanisms of localization control are poorly understood. Here, we use combinations of transgene complementation and live-cell imaging assays to uncover an evolutionarily conserved and essential localization determinant in the DOCK-GEF named SPIKE1. The SPIKE1-DHR3 domain is sufficient for organelle association in vivo, and displays a complicated lipid-binding selectivity for both phospholipid head groups and fatty acid chain saturation. SPIKE1-DHR3 is predicted to adopt a C2-domain structure and functions as part of a tandem C2 array that enables reversible clustering at the cell apex. This work provides mechanistic insight into how DOCK GEFs sense compositional and biophysical membrane properties at the interface of two organelle systems.

Recombinant MBP-pσ1 expressed in soybean seeds delays onset and reduces developing disease in an animal model of multiple sclerosis.

It is estimated that multiple sclerosis (MS) affects over 2.8 million people worldwide, with a prevalence that is expected to continue growing over time. Unfortunately, there is no cure for this autoimmune disease. For several decades, antigen-specific treatments have been used in animal models of experimental autoimmune encephalomyelitis (EAE) to demonstrate their potential for suppressing autoimmune responses. Successes with preventing and limiting ongoing MS disease have been documented using a wide variety of myelin proteins, peptides, autoantigen-conjugates, and mimics when administered by various routes. While those successes were not translatable in the clinic, we have learned a great deal about the roadblocks and hurdles that must be addressed if such therapies are to be useful. Reovirus sigma1 protein (pσ1) is an attachment protein that allows the virus to target M cells with high affinity. Previous studies showed that autoantigens tethered to pσ1 delivered potent tolerogenic signals and diminished autoimmunity following therapeutic intervention. In this proof-of-concept study, we expressed a model multi-epitope autoantigen (human myelin basic protein, MBP) fused to pσ1 in soybean seeds. The expression of chimeric MBP-pσ1 was stable over multiple generations and formed the necessary multimeric structures required for binding to target cells. When administered to SJL mice prophylactically as an oral therapeutic, soymilk formulations containing MBP-pσ1 delayed the onset of clinical EAE and significantly reduced developing disease. These results demonstrate the practicality of soybean as a host for producing and formulating immune-modulating therapies to treat autoimmune diseases.

Local Auxin Biosynthesis Is a Key Regulator of Plant Development.

Auxin is a major phytohormone that controls numerous aspects of plant development and coordinates plant responses to the environment. Morphogenic gradients of auxin govern cell fate decisions and underlie plant phenotypic plasticity. Polar auxin transport plays a central role in auxin maxima generation. The discovery of the exquisite spatiotemporal expression patterns of auxin biosynthesis genes of the WEI8/TAR and YUC families suggested that local auxin production may contribute to the formation of auxin maxima. Herein, we systematically addressed the role of local auxin biosynthesis in plant development and responses to the stress phytohormone ethylene by manipulating spatiotemporal patterns of WEI8. Our study revealed that local auxin biosynthesis and transport act synergistically and are individually dispensable for root meristem maintenance. In contrast, flower fertility and root responses to ethylene require local auxin production that cannot be fully compensated for by transport in the generation of morphogenic auxin maxima.

Local auxin sources orient the apical-basal axis in Arabidopsis embryos.

Establishment of the embryonic axis foreshadows the main body axis of adults both in plants and in animals, but underlying mechanisms are considered distinct. Plants utilize directional, cell-to-cell transport of the growth hormone auxin to generate an asymmetric auxin response that specifies the embryonic apical-basal axis. The auxin flow directionality depends on the polarized subcellular localization of PIN-FORMED (PIN) auxin transporters. It remains unknown which mechanisms and spatial cues guide cell polarization and axis orientation in early embryos. Herein, we provide conceptually novel insights into the formation of embryonic axis in Arabidopsis by identifying a crucial role of localized tryptophan-dependent auxin biosynthesis. Local auxin production at the base of young embryos and the accompanying PIN7-mediated auxin flow toward the proembryo are required for the apical auxin response maximum and the specification of apical embryonic structures. Later in embryogenesis, the precisely timed onset of localized apical auxin biosynthesis mediates PIN1 polarization, basal auxin response maximum, and specification of the root pole. Thus, the tight spatiotemporal control of distinct local auxin sources provides a necessary, non-cell-autonomous trigger for the coordinated cell polarization and subsequent apical-basal axis orientation during embryogenesis and, presumably, also for other polarization events during postembryonic plant life.

A loss-of-function mutation in the nucleoporin AtNUP160 indicates that normal auxin signalling is required for a proper ethylene response in Arabidopsis.

As part of a continuing effort to elucidate mechanisms that regulate the magnitude of ethylene signalling, an Arabidopsis mutant with an enhanced ethylene response was identified. Subsequent characterization of this loss-of-function mutant revealed severe hypocotyl shortening in the presence of saturating ethylene along with increased expression in leaves of a subset of ethylene-responsive genes. It was subsequently determined by map-based cloning that the mutant (sar1-7) represents a loss-of-function mutation in the previously described nucleoporin AtNUP160 (At1g33410, SAR1). In support of previously reported results, the sar1-7 mutant partially restored auxin responsiveness to roots of an rce1 loss-of-function mutant, indicating that AtNUP160/SAR1 is required for proper expression of factors responsible for the repression of auxin signalling. Analysis of arf7-1/sar1-7 and arf19-1/sar1-7 double mutants revealed that mutations affecting either ARF7 or ARF19 function almost fully blocked manifestation of the sar1-7-dependent ethylene hypersensitivity phenotype, suggesting that ARF7- and ARF19-mediated auxin signalling is responsible for regulating the magnitude of and/or competence for the ethylene response in Arabidopsis etiolated hypocotyls. Consistent with this, addition of auxin to ethylene-treated seedlings resulted in severe hypocotyl shortening, reminiscent of that seen for other eer (enhanced ethylene response) mutants, suggesting that auxin functions in part synergistically with ethylene to control hypocotyl elongation and other ethylene-dependent phenomena.

The Arabidopsis YUCCA1 flavin monooxygenase functions in the indole-3-pyruvic acid branch of auxin biosynthesis.

The effects of auxins on plant growth and development have been known for more than 100 years, yet our understanding of how plants synthesize this essential plant hormone is still fragmentary at best. Gene loss- and gain-of-function studies have conclusively implicated three gene families, CYTOCHROME P450 79B2/B3 (CYP79B2/B3), YUCCA (YUC), and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE-RELATED (TAA1/TAR), in the production of this hormone in the reference plant Arabidopsis thaliana. Each of these three gene families is believed to represent independent routes of auxin biosynthesis. Using a combination of pharmacological, genetic, and biochemical approaches, we examined the possible relationships between the auxin biosynthetic pathways defined by these three gene families. Our findings clearly indicate that TAA1/TARs and YUCs function in a common linear biosynthetic pathway that is genetically distinct from the CYP79B2/B3 route. In the redefined TAA1-YUC auxin biosynthetic pathway, TAA1/TARs are required for the production of indole-3-pyruvic acid (IPyA) from Trp, whereas YUCs are likely to function downstream. These results, together with the extensive genetic analysis of four pyruvate decarboxylases, the putative downstream components of the TAA1 pathway, strongly suggest that the enzymatic reactions involved in indole-3-acetic acid (IAA) production via IPyA are different than those previously postulated, and a new and testable model for how IAA is produced in plants is needed.

The eer5 mutation, which affects a novel proteasome-related subunit, indicates a prominent role for the COP9 signalosome in resetting the ethylene-signaling pathway in Arabidopsis.

An Arabidopsis mutant, eer5-1, which has an enhanced ethylene response in etiolated seedlings, including hypersensitivity and extreme exaggeration of response to ethylene, was isolated and characterized. As with other identified eer mutants, the enhanced response phenotype of eer5-1 was correlated with failure to induce appropriately a subset of ethylene-regulated genes, suggesting that proper ethylene-responsive gene expression is necessary for resetting the ethylene response pathway. eer5-1 represents a mutation that causes an amino acid substitution in a previously uncharacterized gene, which encodes a protein with a PAM [proteasome COP9 initiation factor (PCI/PINT)-associated module] domain similar to those found in components of the COP9 signalosome (CSN). Genetic analysis shows that manifestation of the eer5 mutant phenotype is solely dependent on ethylene signaling, as the ein2-5 eer5-1 double mutant was indistinguishable from ein2-5 in the presence of saturating ethylene concentrations. In contrast, the ein3-1 eer5-1 double mutant displayed characteristics of an enhanced ethylene response, and this suggests that EER5 regulates ethylene signaling independently of EIN3. Analysis of the EER5 protein indicates that it interacts with the C-terminus of EIN2 and with the CSN, suggesting that EER5 serves as a bridge between EIN2 and the modification or degradation of target proteins, including a proposed group of transcriptional repressors, as part of a resetting mechanism during or following ethylene signaling.

Arabidopsis enhanced ethylene response 4 encodes an EIN3-interacting TFIID transcription factor required for proper ethylene response, including ERF1 induction.

eer4 was isolated as an Arabidopsis mutant with an extreme response to ethylene in dark-grown seedlings that was also found to have partial ethylene insensitivity at the level of ethylene-dependent gene expression, including ERF1. Subsequent cloning of eer4 revealed an inappropriate stop codon in a previously uncharacterized TFIID-interacting transcription factor homologous to human TAF12 and yeast TAF61. Genetic and pharmacological analysis demonstrated that the eer4 phenotype is strictly ethylene dependent in seedlings, yet a double mutant with the partially ethylene-insensitive Arabidopsis mutant, ein3-1, had restored ethylene responsiveness, indicating that eer4 also regulates a previously unknown resetting or dampening mechanism for the ethylene signalling pathway. Consistent with the absolute requirement of EER4 for ERF1 expression, biochemical analysis showed that EER4 is localized to the nucleus where it probably recruits EIN3 and probably other transcription factors along with components of the TFIID complex for expression of a subset of genes required for either manifestation or subsequent dampening of the response to ethylene.