The family of aquaporins, also called water channels or major intrinsic proteins, is characterized by six transmembrane domains that together facilitate the transport of water and a variety of low molecular weight solutes. They are found in all domains of life, but show their highest diversity in plants. Numerous studies identified aquaporins as important targets for improving plant performance under drought stress. The phylogeny of aquaporins is well established based on model species like Arabidopsis thaliana, which can be used as a template to investigate aquaporins in other species. In this study we comprehensively identified aquaporin encoding genes in tomato (Solanum lycopersicum), which is an important vegetable crop and also serves as a model for fleshy fruit development. We found 47 aquaporin genes in the tomato genome and analyzed their structural features. Based on a phylogenetic analysis of the deduced amino acid sequences the aquaporin genes were assigned to five subfamilies (PIPs, TIPs, NIPs, SIPs and XIPs) and their substrate specificity was assessed on the basis of key amino acid residues. As ESTs were available for 32 genes, expression of these genes was analyzed in 13 different tissues and developmental stages of tomato. We detected tissue-specific and development-specific expression of tomato aquaporin genes, which is a first step towards revealing the contribution of aquaporins to water and solute transport in leaves and during fruit development.

The mobility of sugars between source and sink tissues in plants depends on sugar transport proteins. Studying the corresponding genes allows the manipulation of the sink strength of developing fruits, thereby improving fruit quality for human consumption. Tomato (Solanum lycopersicum) is both a major horticultural crop and a model for the development of fleshy fruits. In this article we provide a comprehensive inventory of tomato sugar transporters, including the SUCROSE TRANSPORTER family, the SUGAR TRANSPORTER PROTEIN family, the SUGAR FACILITATOR PROTEIN family, the POLYOL/MONOSACCHARIDE TRANSPORTER family, the INOSITOL TRANSPORTER family, the PLASTIDIC GLUCOSE TRANSLOCATOR family, the TONOPLAST MONOSACCHARIDE TRANSPORTER family and the VACUOLAR GLUCOSE TRANSPORTER family. Expressed sequence tag (EST) sequencing and phylogenetic analyses established a nomenclature for all analyzed tomato sugar transporters. In total we identified 52 genes in tomato putatively encoding sugar transporters. The expression of 29 sugar transporter genes in vegetative tissues and during fruit development was analyzed. Several sugar transporter genes were expressed in a tissue- or developmental stage-specific manner. This information will be helpful to better understand source to sink movement of photoassimilates in tomato. Identification of fruit-specific sugar transporters might be a first step to find novel genes contributing to tomato fruit sugar accumulation.

Plasma membrane (PM) H+-ATPase in guard cells is activated by phosphorylation of the penultimate residue, threonine (Thr), in response to blue and red light, promoting stomatal opening. Previous in vitro biochemical investigation suggested that Mg2+- and Mn2+-dependent membrane-localized type 2C protein phosphatase (PP2C)-like activity mediates dephosphorylation of PM H+-ATPase in guard cells. PP2C clade D (PP2C.D) was later demonstrated to be involved in PM H+-ATPase dephosphorylation during auxin-induced cell expansion in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether PP2C.D phosphatases are involved in PM H+-ATPase dephosphorylation in guard cells. Transient expression experiments using Arabidopsis mesophyll cell protoplasts revealed that all PP2C.D isoforms dephosphorylate the endogenous PM H+-ATPase. We further analyzed PP2C.D6/8/9, which display higher expression levels than other isoforms in guard cells, observing that pp2c.d6, pp2c.d8 and pp2c.d9 single mutants showed similar light-induced stomatal opening and phosphorylation status of PM H+-ATPase in guard cells as Col-0. By contrast, the pp2c.d6/9 double mutant displayed wider stomatal apertures and greater PM H+-ATPase phosphorylation in response to blue light, but delayed dephosphorylation of PM H+-ATPase in guard cells; the pp2c.d6/8/9 triple mutant showed similar phenotypes to those of the pp2c.d6/9 double mutant. Taken together, these results indicate that PP2C.D6 and PP2C.D9 redundantly mediate PM H+-ATPase dephosphorylation in guard cells. Curiously, unlike auxin-induced cell expansion in seedlings, auxin had no effect on the phosphorylation status of PM H+-ATPase in guard cells.

Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic blistering disorder, caused by mutations in the COL7A1 gene encoding for type VII collagen (C7) protein. These mutations result amongst other complications in fragility of the skin and mucosal membranes. RNA exon-skipping is the modulation of splicing of a pre-mRNA in order to prevent inclusion of a targeted exon into the mRNA. Many RDEB patients harbour a mutation in exon 73 of the COL7A1 gene. We aimed to identify an antisense oligonucleotide (AON) that would exclude the in-frame exon 73 from the COL7A1 mRNA. Exon-skipping in COL7A1 can result in a slightly shortened C7 protein. QR-313, a fully phosphorothioated and 2'O-methylated AON, composed of 21 bases was selected. This sequence is optimized for length, sequence, lack of immunogenicity and optimal manufacturability. We have tested the effectiveness of QR-313 using multiple cell-types, including primary wild type fibroblasts, (modified) HaCaT cells and dermal fibroblasts compound heterozygous RDEB patient, harbouring a mutation in exon 73. These cells were examined for their ability to induce exon 73 skipping from the COL7A1 mRNA using an in vitro transfection system. Products were analysed using PCR and results demonstrate that QR-313 efficiently excludes exon 73 from the COL7A1 mRNA. Secondly, we have assessed the RNA exon skip potential of QR-313 in human skin equivalents (HSEs). HSEs are composed of a dermal layer, harbouring fibroblasts, with a differentiated epidermal layer on top. Before application of QR-313, HSEs were wounded by partial removal of the epidermis, to mimic the blistered skin of RDEB patients. Topical application of QR-313 in a carbomer-based hydrogel for 24-48 hrs, resulted in the exclusion of exon 73 from the COL7A1 mRNA in dermal fibroblasts and epidermal keratinocytes. QR-313 is a potential novel RNA oligonucleotide that could treat RDEB due to exon 73 mutations by exon-skipping.

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We previously identified increased involucrin (IVL) expression for human skin barrier evolution that arose out-of-Africa. This led us to re-examine the function of involucrin in both adult and newborn Ivl-/-mice. We investigated the inflammatory responses in adult mice using the MC903 (vitamin D agonist) inducible model for atopic dermatitis. Unexpectedly, Ivl-/-mice exhibited reduced ear thickness compared to WT mice (p<0.001). Underlying this decreased ear skin inflammation was a comparative decrease in thymic stromal lymphopoietin (Tslp) expression in Ivl-/-treated ears. Flow cytometry analysis to determine innate and adaptive immune cell phenotypes identified a notable decrease in CD4+ T cell infiltrate in Ivl-/-treated ears (p<0.05). We investigated a potential mechanism for the reduced MC903-induced inflammation and identified reduced vitamin D receptor (Vdr) expression in Ivl-/-versus WT skin. Thus far, we have identified a new phenotype for Ivl-/-mice with reduced Vdr-mediated inflammation and decreased adaptive CD4+ T cell response as a result of decreased Vdr. We further examined the impact of involucrin deficiency in the skin using a comprehensive multi-omics approach (ATAC-seq, RNA-seq, and LC/MS proteomics) to determine chromatin accessibility, transcriptomic, and proteomic changes in Ivl-/-and WT newborn epidermis (q<0.05). Involucrin was identified at the intersection of all three datasets thus demonstrating the validity and rigor of the approach. Five potential targets at the intersect of ATAC-seq differentially accessible regions and LC/MS differentially expressed proteins were determined. Of most interest, Csnk1e, known to regulate circadian clock, was found to have less accessible chromatin and reduced protein expression. DEXseq analysis identified a bias for Csnk1e alternative transcripts in Ivl-/-mice. Together our findings reveal a functional role for the evolutionarily selected involucrin to regulate VDR-mediated inflammation and potentially for the circadian response in the skin.

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