Michael Sauer

In eukaryotes, EPSINs are Epsin N-terminal Homology (ENTH) domain-containing proteins that serve as monomeric clathrin adaptors at the plasma membrane (PM) or the trans-Golgi Network (TGN)/early endosomes (EE). The model plant Arabidopsis thaliana encodes for seven ENTH proteins, of which so far, only AtEPSIN1 (AtEPS1) and MODIFIED TRANSPORT TO THE VACUOLE1 (AtMTV1) localize to the TGN/EE and contribute to cargo trafficking to both the cell surface and the vacuole. However, relatively little is known about role(s) of any plant EPSIN in governing physiological responses. We have recently shown that AtEPS1 is a positive modulator of plant immune signaling and pattern-triggered immunity against flagellated Pseudomonas syringae pv. tomato (Pto) DC3000 bacteria. In eps1 mutants, impaired immune responses correlate with reduced accumulation of the receptor FLAGELLIN SENSING2 (AtFLS2) and the convergent immune co-receptor BRASSINOSTEROID INSENTIVE1-ASSOCIATED RECEPTOR KINASE1 (AtBAK1) in the PM. Here, we report that in contrast to AtEPS1, the TGN/EE-localized AtMTV1 did not contribute significantly to immunity against pathogenic Pto DC3000 bacteria. We also compared the amino acid sequences, peptide motif structures and in silico tertiary structures of the ENTH domains of AtEPS1 and AtMTV1 in more detail. We conclude that despite sharing the classical tertiary alpha helical ENTH-domain structure and clathrin-binding motifs, the overall low amino acid identity and differences in peptide motifs may explain their role(s) in trafficking of some of the same as well as distinct cargo components to their site of function, with the latter potentially contributing to differences in physiological responses.

This method determines if two fluorescently labeled proteins are in close proximity to each other in situ. It is an alternative to commonly used co-localization assays and is based on measuring distances between pairs of objects representative of the two proteins. It makes use of a relatively recently developed ImageJ plugin called DiAna, which employs semi-automated object recognition and subsequent distance analysis of the recognized objects. The advantages of this method are that it is largely independent of the actual pixel intensity values, quite robust against background noise, and not reliant on arbitrarily set intensity threshold values. We present here a use case for the DiAna plugin in the context of plant cells with fluorescently labeled subcellular structures, such as proteins associated with the plant Golgi apparatus. Key words Distance analysis Quantitative co-localization Quantitative imaging Object recognition Golgi proteins Subcellular imaging AiryScan

Significance: This is a comprehensive study of Epsin-like accessory proteins in Arabidopsis , for which so far only limited data are available. We identify two members as major accessory proteins at the TGN, controlling vacuolar and a subset of secretory transport. By interaction studies with AP complexes, genetic interactions, and enhanced-resolution microscopy, our work reveals the existence of two independent, parallel transport pathways, which originate from spatially separate regions at the TGN. Our study thus provides strong molecular, cellular, and genetic evidence for spatially, functionally, and molecularly separate subdomains of the plant TGN.

Significance: Vacuoles play crucial roles in plant growth and adaptation to the environment. However, the mechanisms responsible for transporting membranes and contents to plant vacuoles remain largely uncharacterized, and the pathways and compartments involved are not fully charted. We report on the characterization of 17 vacuolar trafficking mutants that define a set of 13 cellular factors involved in transport of soluble vacuolar proteins in Arabidopsis thaliana . We show that two of these factors, the GARP tethering complex subunits VPS51 and VPS54, reside in an ER- and microtubule-associated compartment (EMAC) and that VPS51 interacts with the motor domain of kinesins, revealing an unknown compartment of the vacuolar pathway and suggesting that microtubules and kinesins participate in vacuolar trafficking in plants.

The phytohormone auxin influences virtually all aspects of plant growth and development. Auxin transport across membranes is facilitated by, among other proteins, members of the PIN-FORMED (PIN) and the structurally similar PIN-LIKES (PILS) families, which together govern directional cell-to-cell transport and intracellular accumulation of auxin. Canonical PIN proteins, which exhibit a polar localization in the plasma membrane, determine many patterning and directional growth responses. Conversely, the less-studied non-canonical PINs and PILS proteins, which mostly localize to the endoplasmic reticulum, attenuate cellular auxin responses. Here, and in the accompanying poster, we provide a brief summary of current knowledge of the structure, evolution, function and regulation of these auxin transport facilitators.

Auxin is unique among plant hormones due to its directional transport that is mediated by the polarly distributed PIN auxin transporters at the plasma membrane. The canalization hypothesis proposes that the auxin feedback on its polar flow is a crucial, plant-specific mechanism mediating multiple self-organizing developmental processes. Here, we used the auxin effect on the PIN polar localization in Arabidopsis thaliana roots as a proxy for the auxin feedback on the PIN polarity during canalization. We performed microarray experiments to find regulators of this process that act downstream of auxin. We identified genes that were transcriptionally regulated by auxin in an AXR3/IAA17- and ARF7/ARF19-dependent manner. Besides the known components of the PIN polarity, such as PID and PIP5K kinases, a number of potential new regulators were detected, among which the WRKY23 transcription factor, which was characterized in more detail. Gain- and loss-of-function mutants confirmed a role for WRKY23 in mediating the auxin effect on the PIN polarity. Accordingly, processes requiring auxin-mediated PIN polarity rearrangements, such as vascular tissue development during leaf venation, showed a higher WRKY23 expression and required the WRKY23 activity. Our results provide initial insights into the auxin transcriptional network acting upstream of PIN polarization and, potentially, canalization-mediated plant development. The plant hormone auxin belongs to the major plant-specific developmental regulators. It mediates or modifies almost all aspects of plant life. One of the fascinating features of the auxin action is its directional movement between cells, whose direction can be regulated by auxin signaling itself. This plant-specific feedback regulation has been proposed decades ago and allows for the self-organizing formation of distinct auxin channels shown to be crucial for processes, such as the regular pattern formation of leaf venation, organ formation, and regeneration of plant tissues. Despite the prominent importance of this so called auxin canalization process, the insight into the underlying molecular mechanism is very limited. Here, we identified a number of genes that are transcriptionally regulated and act downstream of the auxin signaling to mediate the auxin feedback on the polarized auxin transport. One of them is the WRKY23 transcription factor that has previously been unsuspected to play a role in this process. Our work provides the first insights into the transcriptional regulation of the auxin canalization and opens multiple avenues to further study this crucial process.

Coordinated cell polarization in developing tissues is a recurrent theme in multicellular organisms. In plants, a directional distribution of the plant hormone auxin is at the core of many developmental programs. A feedback regulation of auxin on the polarized localization of PIN auxin transporters in individual cells has been proposed as a self-organizing mechanism for coordinated tissue polarization, but the molecular mechanisms linking auxin signalling to PIN-dependent auxin transport remain unknown. We used a microarray-based approach to find regulators of the auxin-induced PIN relocation in Arabidopsis thaliana root, and identified a subset of a family of phosphatidylinositol transfer proteins (PITPs), the PATELLINs (PATLs). Here, we show that PATLs are expressed in partially overlapping cell types in different tissues going through mitosis or initiating differentiation programs. PATLs are plasma membrane-associated proteins accumulated in Arabidopsis embryos, primary roots, lateral root primordia and developing stomata. Higher order patl mutants display reduced PIN1 repolarization in response to auxin, shorter root apical meristem, and drastic defects in embryo and seedling development. This suggests that PATLs play a redundant and crucial role in polarity and patterning in Arabidopsis.

The Arabidopsis RPAP2/RTR1 homolog RIMA interacts with IYO and mediates its nuclear accumulation to activate cell differentiation that cannot be reversed by aux