Seeds of Arabidopsis plants expressing dominant-negative AtSKD1 under control of the GL2 promoter show a transparent testa phenotype and a mucilage defect

We have recently shown that overexpression of dominant-negative AtSKD1 versions under control of the trichome and non-root-hair-cell specific GL2 promoter (GL2pro) blocks trafficking of soluble cargo to the vacuole, resulting in its fragmentation and ultimately cell death. GL2pro is also active in the Arabidopsis seeds. When we inspected seeds of the dominant-negative AtSKD1 variants we found two phenotypes. The seeds display a transparent testa phenotype caused by a lack of proanthocyanidin (PA) and do not possess seed coat mucilage. Both phenotypes could be connected by cell death induced by the overexpression of dominant-negative AtSKD1

ESCRT-I Mediates FLS2 Endosomal Sorting and Plant Immunity

The plant immune receptor FLAGELLIN SENSING 2 (FLS2) is present at the plasma membrane and is internalized following activation of its ligand flagellin (flg22). We show that ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT (ESCRT)-I subunits play roles in FLS2 endocytosis in Arabidopsis. VPS37-1 co-localizes with FLS2 at endosomes and immunoprecipitates with the receptor upon flg22 elicitation. Vps37-1 mutants are reduced in flg22-induced FLS2 endosomes but not in endosomes labeled by Rab5 GTPases suggesting a defect in FLS2 trafficking rather than formation of endosomes. FLS2 localizes to the lumen of multivesicular bodies, but this is altered in vps37-1 mutants indicating compromised endosomal sorting of FLS2 by ESCRT-I loss-of-function. VPS37-1 and VPS28-2 are critical for immunity against bacterial infection through a role in stomatal closure. Our findings identify that VPS37-1, and likewise VPS28-2, regulate late FLS2 endosomal sorting and reveals that ESCRT-I is critical for flg22-activated stomatal defenses involved in plant immunity

Analysis of TTG1 function in Arabis alpina

Background: In Arabidopsis thaliana (A. thaliana) the WD40 protein TRANSPARENT TESTA GLABRA1 (TTG1) controls five traits relevant for the adaptation of plants to environmental changes including the production of proanthocyanidin, anthocyanidin, seed coat mucilage, trichomes and root hairs. The analysis of different Brassicaceae species suggests that the function of TTG1 is conserved within the family. Results: In this work, we studied the function of TTG1 in Arabis alpina (A. alpina). A comparison of wild type and two Aattg1 alleles revealed that AaTTG1 is involved in the regulation of all five traits. A detailed analysis of the five traits showed striking phenotypic differences between A. alpina and A. thaliana such that trichome formation occurs also at later stages of leaf development and that root hairs form at non-root hair positions. Conclusions: The evolutionary conservation of the regulation of the five traits by TTG1 on the one hand and the striking phenotypic differences make A. alpina a very interesting genetic model system to study the evolution of TTG1-dependent gene regulatory networks at a functional level

The AAA-type ATPase AtSKD1 contributes to vacuolar maintenance of Arabidopsis thaliana

P>The vacuole is the most prominent organelle of plant cells. Despite its importance for many physiological and developmental aspects of plant life, little is known about its biogenesis and maintenance. Here we show that Arabidopsis plants expressing a dominant-negative version of the AAA (ATPase associated with various cellular activities) ATPase AtSKD1 (SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1) under the control of the trichome-specific GLABRA2 (GL2) promoter exhibit normal vacuolar development in early stages of trichome development. Shortly after its formation, however, the large central vacuole is fragmented and finally disappears completely. Secretion assays with amylase fused to the vacuolar sorting signal of Sporamin show that dominant-negative AtSKD1 inhibits vacuolar trafficking of the reporter that is instead secreted. In addition, trichomes expressing dominant-negative AtSKD1 frequently contain multiple nuclei. Our results suggest that AtSKD1 contributes to vacuolar protein trafficking and thereby to the maintenance of the large central vacuole of plant cells, and might play a role in cell-cycle regulation

Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway

Background: In yeast and mammals, many plasma membrane (PM) proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT) machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. Results: Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. Conclusions: Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route, but it also mediates vacuolar delivery if displayed at the Golgi. In both cases, ubiquitin-tagged proteins travel via early endosomes and multivesicular bodies to the lytic vacuole. This suggests that vacuolar degradation of ubiquitinated proteins is not restricted to PM proteins but might also facilitate the turnover of membrane proteins in the early secretory pathway

FLS2 is transported as luminal cargo of MVBs.

<p>(<i>A</i>) Standard confocal micrographs of FLS2-GFP transgenic leaf epidermal cells transiently expressing RFP-ARA7/RabF2b treated with 10 µM flg22 for 60 min; bar = 10 µm. (<i>B, C</i>) Detail images of co-localizing FLS2-GFP and RFP-ARA7/RabF2b as indicated by white boxes in <i>A</i>; bar = 1 µm. (<i>B</i>) Detail image of normal size MVB. (<i>C</i>) Detail image of the ring-like structure of enlarged MVBs. (<i>D</i>) Confocal micrographs of FLS2-GFP × ARA6/RabF1-RFP transgenic leaf epidermal cells treated with 10 µM flg22 for 45 min followed by 30 µM Wortmannin for 2 h; bar = 10 µm. (<i>E</i>) Detail images of co-localized FLS2-GFP and ARA6/RabF1-RFP at enlarged, ring-like structured MVBs; bar = 1 µm. Transections across endosomes used for fluorescence intensity measurements are indicated by white lines. The histograms show FLS2-GFP, RFP-ARA7/RabF1, and ARA6/RabF1-RFP fluorescent intensities depicted by green and red lines, respectively. Representative images of at least three experiments are shown.</p

Knock-out <i>vps37-1</i> mutants are impaired in immunity.

<p>(<i>A</i>) Four weeks-old plants of the indicated genotypes were surface inoculated with <i>Pto</i> DC3000 and bacterial multiplication was monitored at 0 (4 hrs) and 3 dpi. Shown are mean values +/− SE; n = 6; asterisks indicate significant differences p<0.01 based on ANOVA and Tukey's honestly test. (<i>B</i>) Stomatal apertures were measured following treatments with water (mock), 10 µM flg22, 5 µM ABA for 90 min. Bars represent mean values +/− SE; n>138<268 stomata. All statistical analysis is based on ANOVA and Tukey's honestly test and letters indicate statistical significance of p<0.05. (<i>C</i>) ROS generation in leaf discs of the indicated genotypes triggered by 10 µM flg22 over time. Error bars represent mean values +/− SE; n = 16. All experiments have been repeated at least three times.</p

Flg22-induced endocytosis of FLS2 is compromised in <i>vps37-1</i> mutants but not steady-state ARA6/RabF1 and ARA7/RabF2b endosomal numbers.

<p>(<i>A</i>) High-throughput confocal micrographs show Arabidopsis cotyledon cells of the indicated FLS2-GFP transgenic lines treated with 10 µM flg22 for 80 min. Detail pictures show FLS2-GFP endosomes (indicated with arrows); bar = 10 µm; numbers indicate total endosomes. (<i>B</i>) Immunoblot detection of endogenous FLS2 and transgenic FLS2-GFP protein accumulation in homozygous Col-0 and <i>vps37-1</i> plants. Coomassie brilliant blue (CBB) is used as loading control. (<i>C</i>) Quantification of FLS2-GFP endosomal numbers upon treatment with 10 µM flg22 at the indicated times and genotypes. Error bars represent mean values +/− SE; n>26<83 images. Asterisks indicate statistical significance of p<0.05 (*) and p<0.001 (***). (<i>D</i>) High-throughput confocal micrographs show Arabidopsis cotyledon cells of the indicated ARA6/RabF1-RFP and RFP-ARA7/RabF2b transgenic lines; bar = 10 µm; numbers indicate total endosomes. (<i>E</i>) Quantification of ARA6/RabF1-RFP and RFP-ARA7/RabF2b endosomal numbers. Error bars represent mean values +/− SE; n images. Letters indicate statistical significance of p<0.05. (<i>F</i>) Quantification of the FLS2-GFP fluorescence signal at the plasma membrane upon treatment with 10 µM flg22 at the indicated times and genotypes. Error bars represent mean values +/− SE; n = 50. Letters indicate statistical significance (p<0.05, ANOVA and Tukey's honestly test). These experiments have been repeated three times.</p

A competitive complex formation mechanism underlies trichome patterning on Arabidopsis leaves

Trichome patterning in Arabidopsis serves as a model system for de novo pattern formation in plants. It is thought to typify the theoretical activator–inhibitor mechanism, although this hypothesis has never been challenged by a combined experimental and theoretical approach. By integrating the key genetic and molecular data of the trichome patterning system, we developed a new theoretical model that allows the direct testing of the effect of experimental interventions and in the prediction of patterning phenotypes. We show experimentally that the trichome inhibitor TRIPTYCHON is transcriptionally activated by the known positive regulators GLABRA1 and GLABRA3. Further, we demonstrate by particle bombardment of protein fusions with GFP that TRIPTYCHON and CAPRICE but not GLABRA1 and GLABRA3 can move between cells. Finally, theoretical considerations suggest promoter swapping and basal overexpression experiments by means of which we are able to discriminate three biologically meaningful variants of the trichome patterning model. Our study demonstrates that the mutual interplay between theory and experiment can reveal a new level of understanding of how biochemical mechanisms can drive biological patterning processes