Differential degradation of RNA species by autophagy-related pathways in Arabidopsis.

The plant vacuole recycles proteins and RNA delivered to it by autophagy. In this study, by isolating intact vacuoles from Arabidopsis plants, followed by subsequent RNA purification, and deep sequencing, we provide a comprehensive characterization of Arabidopsis vacuolar RNAome. In the vacuolar RNAome, we detected ribosomal RNAs, transfer RNAs, including those of chloroplast origin, and in addition small RNA types. As autophagy is a main mechanism for the transport of RNA to the vacuole, atg5-1 mutants deficient in autophagy were included in our analysis. We observed severely reduced amounts of most chloroplast-derived RNA species in these mutants. Comparisons with cellular RNA composition provided an indication of possible up-regulation of alternative RNA breakdown pathways. By contrast, vacuolar RNA processing and composition in plants lacking vacuolar ribonuclease 2, involved in cellular RNA homeostasis, only showed minor alterations, possibly because of the presence of further so far unknown vacuolar RNase species. Among the small RNA types, we detected mature miRNAs in all vacuolar preparations but at much lower frequency in atg5-1, raising the possibility of a biological role for vacuolar miRNAs.

Natural genetic variation for expression of a SWEET transporter among wild species of Solanum lycopersicum (tomato) determines the hexose composition of ripening tomato fruit.

The sugar content of Solanum lycopersicum (tomato) fruit is a primary determinant of taste and quality. Cultivated tomato fruit are characterized by near-equimolar levels of the hexoses glucose and fructose, derived from the hydrolysis of translocated sucrose. As fructose is perceived as approximately twice as sweet as glucose, increasing its concentration at the expense of glucose can improve tomato fruit taste. Introgressions of the FgrH allele from the wild species Solanum habrochaites (LA1777) into cultivated tomato increased the fructose-to-glucose ratio of the ripe fruit by reducing glucose levels and concomitantly increasing fructose levels. In order to identify the function of the Fgr gene, we combined a fine-mapping strategy with RNAseq differential expression analysis of near-isogenic tomato lines. The results indicated that a SWEET protein was strongly upregulated in the lines with a high fructose-to-glucose ratio. Overexpressing the SWEET protein in transgenic tomato plants dramatically reduced the glucose levels and increased the fructose : glucose ratio in the developing fruit, thereby proving the function of the protein. The SWEET protein was localized to the plasma membrane and expression of the SlFgr gene in a yeast line lacking native hexose transporters complemented growth with glucose, but not with fructose. These results indicate that the SlFgr gene encodes a plasma membrane-localized glucose efflux transporter of the SWEET family, the overexpression of which reduces glucose levels and may allow for increased fructose levels. This article identifies the function of the tomato Fgr gene as a SWEET transporter, the upregulation of which leads to a modified sugar accumulation pattern in the fleshy fruit. The results point to the potential of the inedible wild species to improve fruit sugar accumulation via sugar transport mechanisms.

Lipoate-Protein Ligase and Octanoyltransferase Are Essential for Protein Lipoylation in Mitochondria of Arabidopsis.

Prosthetic lipoyl groups are required for the function of several essential multienzyme complexes, such as pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase (KGDH), and the glycine cleavage system (glycine decarboxylase [GDC]). How these proteins are lipoylated has been extensively studied in prokaryotes and yeast (Saccharomyces cerevisiae), but little is known for plants. We earlier reported that mitochondrial fatty acid synthesis by ketoacyl-acyl carrier protein synthase is not vital for protein lipoylation in Arabidopsis (Arabidopsis thaliana) and does not play a significant role in roots. Here, we identify Arabidopsis lipoate-protein ligase (AtLPLA) as an essential mitochondrial enzyme that uses octanoyl-nucleoside monophosphate and possibly other donor substrates for the octanoylation of mitochondrial PDH-E2 and GDC H-protein; it shows no reactivity with bacterial and possibly plant KGDH-E2. The octanoate-activating enzyme is unknown, but we assume that it uses octanoyl moieties provided by mitochondrial ß-oxidation. AtLPLA is essential for the octanoylation of PDH-E2, whereas GDC H-protein can optionally also be octanoylated by octanoyltransferase (LIP2) using octanoyl chains provided by mitochondrial ketoacyl-acyl carrier protein synthase to meet the high lipoate requirement of leaf mesophyll mitochondria. Similar to protein lipoylation in yeast, LIP2 likely also transfers octanoyl groups attached to the H-protein to KGDH-E2 but not to PDH-E2, which is exclusively octanoylated by LPLA. We suggest that LPLA and LIP2 together provide a basal protein lipoylation network to plants that is similar to that in other eukaryotes.

Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2.

The vacuolar membrane is involved in solute uptake into and release from the vacuole, which is the largest plant organelle. In addition to inorganic ions and metabolites, large quantities of protons and sugars are shuttled across this membrane. Current models suggest that the proton gradient across the membrane drives the accumulation and/or release of sugars. Recent studies have associated AtSUC4 with the vacuolar membrane. Some members of the SUC family are plasma membrane proton/sucrose symporters. In addition, the sugar transporters TMT1 and TMT2, which are localized to the vacuolar membrane, have been suggested to function in proton-driven glucose antiport. Here we used the patch-clamp technique to monitor carrier-mediated sucrose transport by AtSUC4 and AtTMTs in intact Arabidopsis thaliana mesophyll vacuoles. In the whole-vacuole configuration with wild-type material, cytosolic sucrose-induced proton currents were associated with a proton/sucrose antiport mechanism. To identify the related transporter on one hand, and to enable the recording of symporter-mediated currents on the other hand, we electrophysiologically characterized vacuolar proteins recognized by Arabidopsis mutants of partially impaired sugar compartmentation. To our surprise, the intrinsic sucrose/proton antiporter activity was greatly reduced when vacuoles were isolated from plants lacking the monosaccharide transporter AtTMT1/TMT2. Transient expression of AtSUC4 in this mutant background resulted in proton/sucrose symport activity. From these studies, we conclude that, in the natural environment within the Arabidopsis cell, AtSUC4 most likely catalyses proton-coupled sucrose export from the vacuole. However, TMT1/2 probably represents a proton-coupled antiporter capable of high-capacity loading of glucose and sucrose into the vacuole.

Intracellular localization of VDAC proteins in plants.

Voltage-dependent anion channels (VDACs) are porin-type beta-barrel diffusion pores. They are prominent in the outer membrane of mitochondria and facilitate metabolite exchange between the organelle and the cytosol. Here we studied the subcellular distribution of a plant VDAC-like protein between plastids and mitochondria in green and non-green tissue. Using in vitro studies of dual-import into mitochondria and chloroplasts as well as transient expression of fluorescence-labeled polypeptides, it could be clearly demonstrated that this VDAC isoform targets exclusively to mitochondria and not to plastids. Our results support the idea that plastids evolved a concept of solute exchange with the cytosol different from that of mitochondria.