(R.T., M.M.)The micronutrient zinc is essential for all living organisms, but it is toxic at high concentrations. Here, to understand the effects of excess zinc on plant cells, we performed an iTRAQ (for isobaric tags for relative and absolute quantification)-based quantitative proteomics approach to analyze microsomal proteins from Arabidopsis (Arabidopsis thaliana) roots. Our approach was sensitive enough to identify 521 proteins, including several membrane proteins. Among them, IRT1, an iron and zinc transporter, and FRO2, a ferric-chelate reductase, increased greatly in response to excess zinc. The expression of these two genes has been previously reported to increase under iron-deficient conditions. Indeed, the concentration of iron was significantly decreased in roots and shoots under excess zinc. Also, seven subunits of the vacuolar H + -ATPase (V-ATPase), a proton pump on the tonoplast and endosome, were identified, and three of them decreased significantly in response to excess zinc. In addition, excess zinc in the wild type decreased V-ATPase activity and length of roots and cells to levels comparable to those of the untreated de-etiolated3-1 mutant, which bears a mutation in V-ATPase subunit C. Interestingly, excess zinc led to the formation of branched and abnormally shaped root hairs, a phenotype that correlates with decreased levels of proteins of several root hair-defective mutants. Our results point out mechanisms of growth defects caused by excess zinc in which cross talk between iron and zinc homeostasis and V-ATPase activity might play a central role.

Membrane trafficking in plants is involved in cellular development and the adaptation to various environmental changes. SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) proteins mediate the fusion between vesicles and organelles to facilitate transport cargo proteins in cells. To characterize further the SNARE protein networks in cells, we carried out interactome analysis of SNARE proteins using 12 transgenic Arabidopsis thaliana plants expressing green fluorescent protein (GFP)-tagged Qa-SNAREs (SYP111, SYP121, SYP122, SYP123, SYP132, SYP21, SYP22, SYP31, SYP32, SYP41, SYP42 and SYP43). Microsomal fractions were prepared from each transgenic root, and subjected to immunoprecipitation (IP) using micromagnetic beads coupled to anti-GFP antibodies. To identify Qa-SNARE-interacting proteins, all immunoprecipitated products were then subjected to mass spectrometric (IP-MS) analysis. The IP-MS data revealed not only known interactions of SNARE proteins, but also unknown interactions. The IP-MS results were next categorized by gene ontology analysis. The data revealed that categories of cellular component organization, the cytoskeleton and endosome were enriched in the SYP2, SYP3 and SYP4 groups. In contrast, transporter activity was classified specifically in the SYP132 group. We also identified a novel interaction between SYP22 and VAMP711, which was validated using co-localization analysis with confocal microscopy and IP. Additional novel SNARE-interacting proteins play roles in vesicle transport and lignin biosynthesis, and were identified as membrane microdomain-related proteins. We propose that Qa-SNARE interactomics is useful for understanding SNARE interactions across the whole cell.

Members of the insulin family peptides have conserved roles in the regulation of growth and metabolism in a wide variety of metazoans. The Drosophila genome encodes seven insulin-like peptide genes, dilp1-7, and the most prominent dilps (dilp2, dilp3, and dilp5) are expressed in brain neurosecretory cells known as "insulin-producing cells" (IPCs). Although these dilps are expressed in the same cells, the expression of each dilp is regulated independently. However, the molecular mechanisms that regulate the expression of individual dilps in the IPCs remain largely unknown. Here, we show that Dachshund (Dac), which is a highly conserved nuclear protein, is a critical transcription factor that specifically regulates dilp5 expression. Dac was strongly expressed in IPCs throughout development. dac loss-of-function analyses revealed a severely reduced dilp5 expression level in young larvae. Dac interacted physically with the Drosophila Pax6 homolog Eyeless (Ey), and these proteins synergistically promoted dilp5 expression. In addition, the mammalian homolog of Dac, Dach1/2, facilitated the promoting action of Pax6 on the expression of islet hormone genes in cultured mammalian cells. These observations indicate the conserved role of Dac/Dach in controlling insulin expression in conjunction with Ey/Pax6.