Identification and characterization of vacuolar nucleotide phosphatases and characterization of an engineered catabolic pathway for urea in Arabidopsis thaliana

Abstract

Plants have the remarkable ability to remobilize nutrients by degrading cellular components such as damaged organelles, proteins, and RNA in vacuoles. According to the current model of RNA degradation in vacuoles, the RNA is degraded in three steps. After the transport of the RNA into vacuoles, it is first degraded to 3’-mononucleotides (3'-NMPs) by the intercellular ribonuclease RNS2. The RNA degradation products are then dephosphorylated by vacuolar acid phosphatases (AP), releasing nucleosides. These nucleosides are eventually transported by the Equilibrative Nucleoside Transporter 1 (ENT1) from vacuoles to the cytosol, where they are either degraded or salvaged to nucleotides, thereby either releasing nutrients or conserving metabolic resources. RNS2 and ENT1 were studied and characterized several years ago, but the vacuolar nucleotide phosphatases have not yet been identified. This study focuses on the identification and characterization of the nucleotide-dephosphorylating APs from vacuoles. Three protein families, the Haloacid Dehalogenase IIIB (HADIIIB) family, the Purple Acid Phosphatase (PAP) family, and the Endonuclease (Endo) S1/P1-type nucleases were considered based on their previous identification in the vacuolar proteome or their potential to localize in the vacuole and based on their potential nucleotidase activities. In total 44 candidate proteins were then screened employing a set of selection criteria, including protein conservation in other plants, protein localization, substrate preferences, and expression profiles. Several mutant combinations were generated for the most promising candidates using T-DNA insertion lines and the CRISPR/Cas9 technique. When the final candidates (HADIIIB4 renamed to Vacuolar Nucleoside Phosphate Phosphatase 1 (VNPP1) and PAP26)) were identified, they were fully characterized in vitro and in vivo. The function of VNPP1 and PAP26 in vacuolar mononucleotide dephosphorylation could be demonstrated by the accumulation of 3'-NMPs in the corresponding mutants. Additional tissue-specific vacuolar and non-vacuolar NMP phosphatases were also discovered in the course of this work, opening the door to new research directions for investigating the significance of mononucleotide catabolism in seeds, pollen, and roots.

Nitrogen is one of the most important nutrients for plants. Nitrogen deficiency leads to reduced plant growth and productivity. Urea is the most used nitrogen fertilizer in the world because it has a high nitrogen content and is easy to transport and apply in the field. However, fertilizing exclusively with urea results in reduced growth due to the toxicity of the ammonia. The urea is immediately hydrolysed to ammonia in the roots, the resulting higher ammonia concentration in the roots is toxic, and the plants excrete the excess ammonia back into the soil. In addition, the nitrogen use efficiency of nitrogen fertilizer in general and urea in particular is generally below 50% and needs improvement. Therefore, a biotechnological solution expressing heterologous transgenes in the model plant Arabidopsis thaliana was assessed with the aim to reduce ammonia toxicity for the plant and improve nitrogen use efficiency. For this purpose two enzymes representing an alternative urea catabolism pathway in various bacteria and fungi were investigated. In this system, urea is not immediately hydrolyzed to ammonia as in organisms employing Urease (like plants), but is first carboxylated by a Urea Carboxylase (UC) and the product allophanate is then hydrolysed by and Allophanate Hydrolase (AH) to ammonia and carbon dioxide. The performance transgenic lines expressing this system in the Urease mutant of Arabidopsis under the control of the 35S promoter was investigated. These lines were able to use urea demonstrating that the heterologous enzymes are functional in the plant. Since urea hydrolysis by UC and AH can occur in two steps, additional lines with wild type background (Urease positive) were investigated where the UC was expressed ubiquitously and the AH was expressed only in the shoot and in the light. In these plants ammonia release from allophanate can only occur in the shoot. Such transgenic plants performed significantly better than the wild-type with pure urea nutrition. The transgenic plants had a higher biomass and leaf area, and a higher nitrogen content. The results raise the hope that low nitrogen use efficiency and ammonia toxicity could be tackled with this system also in crop plants.

Pflanzen haben die bemerkenswerte Fähigkeit, Nährstoffe zu remobilisieren, indem sie zelluläre Komponenten wie beschädigte Organellen, Proteine und RNA in den Vakuolen abbauen. Nach dem derzeitigen Modell des RNA-Abbaus in den Vakuolen wird die RNA in drei Schritten abgebaut. Nach dem Transport der RNA in die Vakuolen wird sie zunächst durch die interzelluläre Ribonuklease RNS2 zu 3'-Mononukleotiden (3'-NMPs) katabolisiert. Die RNA-Abbauprodukte werden dann durch saure Vakuolenphosphatasen (AP) dephosphoryliert, wodurch Nukleoside freigesetzt werden. Diese Nukleoside werden schließlich durch den Equilibrative Nucleoside Transporter 1 (ENT1) aus den Vakuolen in das Zytosol transportiert, wo sie entweder weiter abgebaut oder im Salvage-Stoffwechsel zu Nukleotiden recycled werden, wodurch entweder Nährstoffe freigesetzt oder Stoffwechselressourcen geschont werden. RNS2 und ENT1 wurden bereits vor einigen Jahren untersucht und charakterisiert, aber die vakuolären Nukleotidphosphatasen sind noch nicht identifiziert worden. Diese Studie konzentriert sich auf die Identifizierung und Charakterisierung der Nukleotid-dephosphorylierenden APs aus Vakuolen. Es wurden drei Proteinfamilien, die Haloacid Dehalogenase IIIB (HADIIIB)-Familie, die Purple-Acid-Phophatatase (PAP)-Familie und die Endonuklease (Endo) S1/P1-Typ-Nukleasen auf der Grundlage ihrer früheren Identifizierung im Vakuolenproteom oder ihres Potenzials, in der Vakuole zu lokalisieren, und aufgrund ihrer potenziellen Nukleotidase-Aktivitäten in die Untersuchungen einbezogen. Insgesamt wurden 44 Kandidatenproteine anhand einer Reihe von Auswahlkriterien wie Proteinkonservierung in anderen Pflanzen, Proteinlokalisierung, Substratpräferenzen und Expressionsprofilen untersucht. Für die vielversprechendsten Kandidaten wurden mithilfe von T-DNA-Insertionslinien und der CRISPR/Cas9-Technik mehrere Mutantenkombinationen erzeugt. Als die endgültigen Kandidaten (HADIIIB4, umbenannt in Vacuolar Nucleoside Phosphate Phosphatase 1 (VNPP1) und PAP26)) identifiziert waren, wurden diese in vitro und in vivo vollständig charakterisiert. Die Funktion von VNPP1 und PAP26 bei der vakuolären Dephosphorylierung von Mononukleotiden konnte durch die Akkumulation von 3'-NMPs in den entsprechenden Mutanten belegt werden. Weitere gewebespezifische vakuoläre und nicht-vakuoläre NMP-Phosphatasen wurden im Rahmen dieser Arbeit ebenfalls entdeckt, was neue Forschungsrichtungen für die Untersuchung der Bedeutung des Mononukleotid-Katabolismus in Samen, Pollen und Wurzeln eröffnet.