K63-linked ubiquitin chains are a global signal for endocytosis and contribute to selective autophagy in plants.

In contrast to other eukaryotic model organisms, the closely related ubiquitin (Ub)-conjugating enzymes UBC35 and UBC36 are the main sources of K63-linked Ub chains in Arabidopsis.1 Although K63-linked chains have been associated with the regulation of vesicle trafficking, definitive proof for their role in endocytosis was missing. We show that the ubc35 ubc36 mutant has pleiotropic phenotypes related to hormone and immune signaling. Specifically, we reveal that ubc35-1 ubc36-1 plants have altered turnover of integral membrane proteins including FLS2, BRI1, and PIN1 at the plasma membrane. Our data indicates that K63-Ub chains are generally required for endocytic trafficking in plants. In addition, we show that in plants K63-Ub chains are involved in selective autophagy through NBR1, the second major pathway delivering cargoes to the vacuole for degradation. Similar to autophagy-defective mutants, ubc35-1 ubc36-1 plants display an accumulation of autophagy markers. Moreover, autophagy receptor NBR1 interacts with K63-Ub chains, which are required for its delivery to the lytic vacuole.2 Together, we show that K63-Ub chains act as a general signal required for the two main pathways delivering cargo to the vacuole and thus, to maintain proteostasis.

Sulfur Deficiency Increases Phosphate Accumulation, Uptake, and Transport in Arabidopsis thaliana.

Recent studies have shown various metabolic and transcriptomic interactions between sulfur (S) and phosphorus (P) in plants. However, most studies have focused on the effects of phosphate (Pi) availability and P signaling pathways on S homeostasis, whereas the effects of S availability on P homeostasis remain largely unknown. In this study, we investigated the interactions between S and P from the perspective of S availability. We investigated the effects of S availability on Pi uptake, transport, and accumulation in Arabidopsis thaliana grown under sulfur sufficiency (+S) and deficiency (-S). Total P in shoots was significantly increased under -S owing to higher Pi accumulation. This accumulation was facilitated by increased Pi uptake under -S. In addition, -S increased root-to-shoot Pi transport, which was indicated by the increased Pi levels in xylem sap under -S. The -S-increased Pi level in the xylem sap was diminished in the disruption lines of PHT1;9 and PHO1, which are involved in root-to-shoot Pi transport. Our findings indicate a new aspect of the interaction between S and P by listing the increased Pi accumulation as part of -S responses and by highlighting the effects of -S on Pi uptake, transport, and homeostasis.

Sulfur Deficiency-Induced Glucosinolate Catabolism Attributed to Two ß-Glucosidases, BGLU28 and BGLU30, is Required for Plant Growth Maintenance under Sulfur Deficiency.

Sulfur (S) is an essential element for plants, and S deficiency causes severe growth retardation. Although the catabolic process of glucosinolates (GSLs), the major S-containing metabolites specific to Brassicales including Arabidopsis, has been recognized as one of the S deficiency (-S) responses in plants, the physiological function of this metabolic process is not clear. Two ß-glucosidases (BGLUs), BGLU28 and BGLU30, are assumed to be responsible for this catabolic process as their transcript levels were highly upregulated by -S. To clarify the physiological function of BGLU28 and BGLU30 and their roles in GSL catabolism, we analyzed the accumulation of GSLs and other S-containing compounds in the single and double mutant lines of BGLU28 and BGLU30 and in wild-type plants under different S conditions. GSL levels were highly increased, while the levels of sulfate, cysteine, glutathione and protein were decreased in the double mutant line of BGLU28 and BGLU30 (bglu28/30) under -S. Furthermore, transcript level of Sulfate Transporter1;2, the main contributor of sulfate uptake from the environment, was increased in bglu28/30 mutants under -S. With these metabolic and transcriptional changes, bglu28/30 mutants displayed obvious growth retardation under -S. Overall, our results indicate that BGLU28 and BGLU30 are required for -S-induced GSL catabolism and contribute to sustained plant growth under -S by recycling sulfate to primary S metabolism.

Effects of Thiosulfate as a Sulfur Source on Plant Growth, Metabolites Accumulation and Gene Expression in Arabidopsis and Rice.

Plants are considered to absorb sulfur from their roots in the form of sulfate. In bacteria like Escherichia coli, thiosulfate is a preferred sulfur source. It is converted into cysteine (Cys). This transformation consumes less NADPH and ATP than sulfate assimilation into Cys. In Saccharomyces cerevisiae, thiosulfate promoted growth more than sulfate. In the present study, the availability of thiosulfate, the metabolite transformations and gene expressions it induces were investigated in Arabidopsis and rice as model dicots and monocots, respectively. In Arabidopsis, the thiosulfate-amended plants had lower biomass than those receiving sulfate when sulfur concentrations in the hydroponic medium were above 300 µM. In contrast, rice biomass was similar for plants raised on thiosulfate and sulfate at 300 µM sulfur. Therefore, both plants can use thiosulfate but it is a better sulfur source for rice. In both plants, thiosulfate levels significantly increased in roots following thiosulfate application, indicating that the plants absorbed thiosulfate into their root cells. Thiosulfate is metabolized in plants by a different pathway from that used for sulfate metabolism. Thiosulfate increases plant sulfide and cysteine persulfide levels which means that plants are in a more reduced state with thiosulfate than with sulfate. The microarray analysis of Arabidopsis roots revealed that 13 genes encoding Cys-rich proteins were upregulated more with thiosulfate than with sulfate. These results together with those of the widely targeted metabolomics analysis were used to proposes a thiosulfate assimilation pathway in plants.

Glucosinolate Distribution in the Aerial Parts of sel1-10, a Disruption Mutant of the Sulfate Transporter SULTR1;2, in Mature Arabidopsis thaliana Plants.

Plants take up sulfur (S), an essential element for all organisms, as sulfate, which is mainly attributed to the function of SULTR1;2 in Arabidopsis. A disruption mutant of SULTR1;2, sel1-10, has been characterized with phenotypes similar to plants grown under sulfur deficiency (-S). Although the effects of -S on S metabolism were well investigated in seedlings, no studies have been performed on mature Arabidopsis plants. To study further the effects of -S on S metabolism, we analyzed the accumulation and distribution of S-containing compounds in different parts of mature sel1-10 and of the wild-type (WT) plants grown under long-day conditions. While the levels of sulfate, cysteine, and glutathione were almost similar between sel1-10 and WT, levels of glucosinolates (GSLs) differed between them depending on the parts of the plant. GSLs levels in the leaves and stems were generally lower in sel1-10 than those in WT. However, sel1-10 seeds maintained similar levels of aliphatic GSLs to those in WT plants. GSL accumulation in reproductive tissues is likely to be prioritized even when sulfate supply is limited in sel1-10 for its role in S storage and plant defense.