TY - BOOK

T1 - Identification and characterization of vacuolar nucleotide phosphatases and characterization of an engineered catabolic pathway for urea in Arabidopsis thaliana

AU - Firdoos, Nabila

N1 - Doctoral thesis

PY - 2023

Y1 - 2023

N2 - 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.

AB - 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.

U2 - 10.15488/13297

DO - 10.15488/13297

M3 - Doctoral thesis

CY - Hannover

ER -