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Despite serving as a major inorganic nitrogen source for plants, ammonium causes toxicity at elevated concentrations, inhibiting root elongation early on. While previous studies have shown that ammonium-inhibited root development relates to ammonium uptake and formation of reactive oxygen species (ROS) in roots, it remained open which mechanisms are underlying the repression of root growth and how plants cope with this inhibitory effect of ammonium. Here, we demonstrate that ammonium-induced apoplastic acidification co-localizes with Fe precipitation and hydrogen peroxide (H2O2) accumulation along the stele of the elongation and differentiation zone in root tips, indicating Fe-dependent ROS formation. By screening ammonium sensitivity in T-DNA insertion lines of ammonium-responsive genes, we identified PDX1.1, which is upregulated by ammonium in the root stele and catalyzes biosynthesis de novo of vitamin B6. Root growth of pdx1.1 mutants is hypersensitive to ammonium, while chemical complementation or overexpression of PDX1.1 restores root elongation. This salvage strategy requires non-phosphorylated forms of vitamin B6 that are able to quench reactive molecular oxygen species and rescue root growth from ammonium inhibition. We propose PDX1.1-mediated synthesis of non-phosphorylated B6 vitamers as a primary strategy to protect roots from ammonium-dependent ROS formation.

To survive and proliferate in diverse environments with varying climate and nutrient availability, plants modulate their metabolism. Achieving a balance between carbon (C) and nitrogen (N) use such that growth and defense mechanisms can be appropriately controlled is critical for plant fitness. The identification of factors that regulate C/N utilization in plants can make a significant contribution to optimization of plant health. Here we show that pyridox(am)ine 5’-phosphate oxidase (PDX3), which regulates vitamin B6homeostasis, influences C/N balance. The B6vitamer imbalance resulting from loss of PDX3 leads to over-accumulation of nitrogenous compounds. A combination of increased glutamate dehydrogenase activity, impairment in the photorespiratory cycle and inappropriate use of endogenous ammonium fuel the metabolic imbalance. Growth at elevated CO2levels further exacerbates thepdx3phenotypes. Interestingly, serine supplementation rescues growth under high CO2likely bypassing the phosphorylated pathway of biosynthesis suggesting that this amino acid is an important commodity. We show that PDX3 function appears dispensable upon thermomorphogenesis, a condition that favors C metabolism. Furthermore, while a low ammonium to nitrate ratio likely accounts for overstimulation of salicylic acid (SA) defense responses inpdx3lines that compromises growth, a basal level of SA protects against loss of PDX3 biochemical function. Overall, the study highlights environmental scenarios where vitamin B6homeostasis, as managed by the salvage pathway enzyme PDX3, is critical and provides insight into how plants reprogram their metabolism under such conditions.

Triphosphate tunnel metalloenzymes (TTMs) are found in all biological kingdoms and have been characterized in microorganisms and animals. Members of the TTM family already characterized have divergent biological functions and act on a range of triphosphorylated substrates (RNA, thiamine triphosphate, inorganic polyphosphate). TTM proteins in plants have received considerably less attention and are unique in that some homologs harbor additional domains including a P-loop kinase and transmembrane domain. Here we report on structural and functional aspects of the multimodular TTM1 and TTM2 of Arabidopsis thaliana. Tissue and cellular microscopy studies show that both AtTTM1 and AtTTM2 are expressed in actively dividing (meristem) tissue and are tail-anchored proteins at the outer mitochondrial membrane - mediated by the single transmembrane domain at the C-terminus, supporting earlier studies. Crystal structures of AtTTM1 in the presence and absence of a non-hydrolyzable ATP analog reveal a catalytically incompetent TTM tunnel domain tightly interacting with the P-loop kinase domain that is locked in an inactive conformation. Structural comparison reveals that a helical hairpin may facilitate movement of the TTM domain thereby activating the kinase. Genetic studies show that AtTTM2 is important for the developmental transition from the vegetative to the reproductive phase in Arabidopsis, whereas its closest paralog AtTTM1 is not. Rational design of mutations based on the 3D structure demonstrates that both the P-loop kinase and TTM tunnel modules of AtTTM2 are required for the developmental switch.

Triphosphate tunnel metalloenzymes (TTMs) are found in all biological kingdoms and have been characterized in microorganisms and animals. Members of the TTM family have divergent biological functions and act on a range of triphosphorylated substrates (RNA, thiamine triphosphate, and inorganic polyphosphate). TTMs in plants have received considerably less attention and are unique in that some homologs harbor additional domains including a P-loop kinase and transmembrane domain. Here, we report on structural and functional aspects of the multimodular TTM1 and TTM2 of Arabidopsis thaliana. Our tissue and cellular microscopy studies show that both AtTTM1 and AtTTM2 are expressed in actively dividing (meristem) tissue and are tail-anchored proteins at the outer mitochondrial membrane, mediated by the single C-terminal transmembrane domain, supporting earlier studies. In addition, we reveal from crystal structures of AtTTM1 in the presence and absence of a nonhydrolyzable ATP analog a catalytically incompetent TTM tunnel domain tightly interacting with the P-loop kinase domain that is locked in an inactive conformation. Our structural comparison indicates that a helical hairpin may facilitate movement of the TTM domain, thereby activating the kinase. Furthermore, we conducted genetic studies to show that AtTTM2 is important for the developmental transition from the vegetative to the reproductive phase in Arabidopsis, whereas its closest paralog AtTTM1 is not. We demonstrate through rational design of mutations based on the 3D structure that both the P-loop kinase and TTM tunnel modules of AtTTM2 are required for the developmental switch. Together, our results provide insight into the structure and function of plant TTM domains.

Knocking out the activity of a pyridoxal 5 '-phosphate phosphatase suppresses the dwarf phenotype of mutants lacking an ATP/ADP transporter and provides insight into vitamin B6 homeostasis.The endoplasmic reticulum (ER)-located ATP/ADP-antiporter (ER-ANT1) occurs specifically in vascular plants. Structurally different transporters mediate energy provision to the ER, but the cellular function of ER-ANT1 is still unknown. Arabidopsis (Arabidopsis thaliana) mutants lacking ER-ANT1 (er-ant1 plants) exhibit a photorespiratory phenotype accompanied by high glycine levels and stunted growth, pointing to an inhibition of glycine decarboxylase (GDC). To reveal whether it is possible to suppress this marked phenotype, we exploited the power of a forward genetic screen. Absence of a so far uncharacterized member of the HaloAcid Dehalogenase (HAD)-like hydrolase family strongly suppressed the dwarf phenotype of er-ant1 plants. Localization studies suggested that the corresponding protein locates to chloroplasts, and activity assays showed that the enzyme dephosphorylates, with high substrate affinity, the B-6 vitamer pyridoxal 5 '-phosphate (PLP). Additional physiological experiments identified imbalances in vitamin B-6 homeostasis in er-ant1 mutants. Our data suggest that impaired chloroplast metabolism, but not decreased GDC activity, causes the er-ant1 mutant dwarf phenotype. We present a hypothesis, setting transport of PLP by ER-ANT1 and chloroplastic PLP dephosphorylation in the cellular context. With the identification of this HAD-type PLP phosphatase, we also provide insight into B-6 vitamer homeostasis.

Despite serving as a major inorganic nitrogen source for plants, ammonium causes toxicity at elevated concentrations, inhibiting root elongation early on. While previous studies have shown that ammonium-inhibited root development relates to ammonium uptake and formation of reactive oxygen species (ROS) in roots, it remains unclear about the mechanisms underlying the repression of root growth and how plants cope with this inhibitory effect of ammonium. In this study, we demonstrate that ammonium-induced apoplastic acidification co-localizes with Fe precipitation and hydrogen peroxide (H

Monoterpenoid indole alkaloids (MIAs) are produced as plant defence compounds. In the medicinal plant Catharanthus roseus, they comprise the anticancer compounds vinblastine and vincristine. The iridoid (monoterpenoid) pathway forms one of the two branches that feed MIA biosynthesis and its activation is regulated by the transcription factor (TF) basic helix-loop-helix (bHLH) iridoid synthesis 1 (BIS1). Here, we describe the identification and characterisation of BIS2, a jasmonate (JA)-responsive bHLH TF expressed preferentially in internal phloem-associated parenchyma cells, which transactivates promoters of iridoid biosynthesis genes and can homodimerise or form heterodimers with BIS1. Stable overexpression of BIS2 in C. roseus suspension cells and transient ectopic expression of BIS2 in C. roseus petal limbs resulted in increased transcript accumulation of methylerythritol-4-phosphate and iridoid pathway genes, but not of other MIA genes or triterpenoid genes. Transcript profiling also indicated that BIS2 expression is part of an amplification loop, as it is induced by overexpression of either BIS1 or BIS2. Accordingly, silencing of BIS2 in C. roseus suspension cells completely abolished the JA-induced upregulation of the iridoid pathway genes and subsequent MIA accumulation, despite the presence of induced BIS1, indicating that BIS2 is essential for MIA production in C. roseus.

Plants make specialized bioactive metabolites to defend themselves against attackers. The conserved control mechanisms are based on transcriptional activation of the respective plant species-specific biosynthetic pathways by the phytohormone jasmonate. Knowledge of the transcription factors involved, particularly in terpenoid biosynthesis, remains fragmentary. By transcriptome analysis and functional screens in the medicinal plant Catharanthus roseus (Madagascar periwinkle), the unique source of the monoterpenoid indole alkaloid (MIA)-type anticancer drugs vincristine and vinblastine, we identified a jasmonate-regulated basic helix-loop-helix (bHLH) transcription factor from clade IVa inducing the monoterpenoid branch of the MIA pathway. The bHLH iridoid synthesis 1 (BIS1) transcription factor transactivated the expression of all of the genes encoding the enzymes that catalyze the sequential conversion of the ubiquitous terpenoid precursor geranyl diphosphate to the iridoid loganic acid. BIS1 acted in a complementary manner to the previously characterized ethylene response factor Octadecanoid derivative-Responsive Catharanthus APETALA2-domain 3 (ORCA3) that transactivates the expression of several genes encoding the enzymes catalyzing the conversion of loganic acid to the downstream MIAs. In contrast to ORCA3, overexpression of BIS1 was sufficient to boost production of high-value iridoids and MIAs in C. roseus suspension cell cultures. Hence, BIS1 might be a metabolic engineering tool to produce sustainably high-value MIAs in C. roseus plants or cultures.