Tuning of SlARF10A dosage by sly-miR160a is critical for auxin-mediated compound leaf and flower development.

miR160 adjusts auxin-mediated development by post-transcriptional regulation of the auxin response factors ARF10/16/17. In tomato, knockdown of miR160 (sly-miR160) suggested that it is required for auxin-driven leaf blade outgrowth, but whether additional developmental events are adjusted by sly-miR160 is not clear. Here, the SlMIR160 genes and the genes of its SlARFs targets were edited by CRISPR/Cas9 resulting in the isolation of loss-of-function mutants. In addition, hypomorphic mutants that accumulate variable reduced levels of sly-miR160a were isolated. We found that the loss-of-function mutants in SlMIR160a (CR-slmir160a-6/7) produced only four wiry leaves, whereas the hypomorphic mutants developed leaves and flowers with graded developmental abnormalities. Phenotypic severity correlated with the upregulation of SlARF10A. Consistent with that, double mutants in SlMIR160a and SlARF10A restored leaf and flower development indicating that over-accumulation of SlARF10A underlay the developmental abnormalities exhibited in the CR-slmir160a mutants. Phenotype severity also correlated with the upregulation of the SHOOT MERISTEMLESS homolog Tomato Knotted 2, which in turn activated the transcription of the cytokinin biosynthesis genes SlIPT2 and SlIPT4. However, no change in Tomato Knotted 2 was detected in the absence of SlARF10A, suggesting that it is upregulated due to auxin signaling suppression by SlARF10A. Knockout of sly-miR160a-targeted SlARFs showed that whereas SlARF10A is indispensable for leaf blade outgrowth and floral organ patterning, the functions of SlARF16A and SlARF17 are redundant. Taken together our results suggest that sly-miR160a promotes blade outgrowth as well as leaf and leaflet initiation and floral organ development through the quantitative regulation of its major target SlARF10A.

Redistribution of CHH Methylation and Small Interfering RNAs across the Genome of Tomato ddm1 Mutants.

In plants, cytosine methylation, an epigenetic mark critical for transposon silencing, is maintained over generations by key enzymes that directly methylate DNA and is facilitated by chromatin remodelers, like DECREASE IN DNA METHYLATION1 (DDM1). Short-interfering RNAs (siRNAs) also mediate transposon DNA methylation through a process called RNA-directed DNA methylation (RdDM). In tomato (Solanum lycopersicum), siRNAs are primarily mapped to gene-rich chromosome arms, and not to pericentromeric regions as in Arabidopsis thaliana Tomato encodes two DDM1 genes. To better understand their functions and interaction with the RdDM pathway, we targeted the corresponding genes via the CRISPR/Cas9 technology, resulting in the isolation of Slddm1a and Slddm1b knockout mutants. Unlike the single mutants, Slddm1a Slddm1b double mutant plants display pleiotropic vegetative and reproductive phenotypes, associated with severe hypomethylation of the heterochromatic transposons in both the CG and CHG methylation contexts. The methylation in the CHH context increased for some heterochromatic transposons and conversely decreased for others localized in euchromatin. We found that the number of heterochromatin-associated siRNAs, including RdDM-specific small RNAs, increased significantly, likely limiting the transcriptional reactivation of transposons in Slddm1a Slddm1b Taken together, we propose that the global production of siRNAs and the CHH methylation mediated by the RdDM pathway are restricted to chromosome arms in tomato. Our data suggest that both pathways are greatly enhanced in heterochromatin when DDM1 functions are lost, at the expense of silencing mechanisms normally occurring in euchromatin.

Response to cytosolic nickel of Slow Vacuolar channels in the hyperaccumulator plant Alyssum bertolonii.

We applied the patch-clamp technique to investigate the transport properties of the Slow Vacuolar (SV) channel identified in leaf vacuoles of Alyssum bertolonii Desv., a nickel hyperaccumulator plant growing in serpentine soil of the northern Apennines (Italy). SV currents recorded in vacuoles from adult plants collected in their natural habitat showed high sensitivity towards cytosolic nickel. Dose-response analyses indicated half-maximal current inhibition at submicromolar concentrations, i.e. up to three orders of magnitude lower than previously reported values from other plant species. The voltage-dependent increase of residual currents at saturating nickel concentrations could be interpreted as relief of channel block by nickel permeation at high positive membrane potentials. Including young plants of A. bertolonii into the study, we found that SV channels from these plants did not display elevated nickel sensitivity. This difference may be related to age-dependent changes in nickel hyperaccumulation of A. bertolonii leaf cells.