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Depiction of B6 vitamers as well as selected purines and pyrimidines. The predominant forms of vitamin B6 are illustrated in the upper and middle rows. The lower row illustrates 4-deoxypyridoxine (4-dPN), a potent antagonist of vitamin B6, as well as cytosine (a pyrimidine) and adenine (a purine). The accepted numbering of the atoms is shown for PLP. PL, pyridoxal; PLP, pyridoxal 5′-phosphate; PM, pyridoxamine; PMP, pyridoxamine 5′-phosphate; PN, pyridoxine; PNP, pyridoxine 5′-phosphate.
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Vitamin B6 is a cofactor for more than 140 essential enzymatic reactions and was recently proposed as a potent antioxidant, playing a role in the photoprotection of plants. De novo biosynthesis of the vitamin has been described relatively recently and is derived from simple sugar precursors as well as glutamine. In addition, the vitamin can be take...
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... such activities have not been investigated in plants. The term vitamin B6 refers to pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM) and their respective 5′ phosphorylated forms (Figure 1). At the chemical structure level, these compounds share a pyridine ring but differ in the substitution at the 4′ position being an alcohol [PN and pyridoxine 5′-phosphate (PNP)], an aldehyde [PL and pyridoxal 5′-phosphate (PLP)] or an amine [PM and pyridoxamine 5′-phosphate (PMP)]. ...
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... ( Gillissen et al., 2000). As vitamin B6 possesses chemical structure homology with pyrimidines (Figure 1), we decided to investigate the ability of the PUP family to transport vitamin B6. Progress in the annotation of the Arabidopsis genome since the work of Gillissen et al. (2000) and a more recent report (Jelesko, 2012) has now led to the establishment of a complete list of the PUP family. ...
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... updated list of the 21 PUP annotations that constitute the family is thus presented in Table S1. A phylogenetic analysis of PUP cDNA and amino acid sequences establishes a classification comprising four different subfamilies ( Figure S1a,b). PUP9, putatively encoding a protein of 45 amino acid residues in length (Table S1) and therefore not compatible with the predicted structure of a functional transporter was omitted from this analysis. ...
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... order to test the ability of members of the PUP protein family to complement the growth phenotype of the MVY30 S. cerevisiae strain, 13 of the PUP cDNAs were reverse transcribed from Arabidopsis (ecotype Columbia) leaf mRNA by reverse transcription polymerase chain reaction (RT-PCR) and cloned into the yeast/bacteria shuttle vector pDR195 ( Rentsch et al., 1995; a list of primers used is given in Table S2). Notably, members of all subfamilies are represented in the complementation analysis (see Figure S1a,b). Each amplified member of the PUP family, with the empty vector as a control, was transformed into the MVY30 strain. ...
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... this rescue is not totally abolished suggesting that secondary transport systems coexist with PUP1 for uptake of the vitamin by leaf cells. Candidates for this activity are PUP2, 3, 4, 14 and 10 (see Figure 1). The lack of observation of a strong phenotype in pup1 lines presumably reflects redundancy within the PUP family of proteins. ...
Citations
... Only a very few transporters of the vitamin are known in eukaryotes and very little literature exists on its transport in organisms [46]. It is known in plant organisms such as Arabidopsis thaliana the only currently known vitamin B6 transporter is the PUP1 transporter of the three vitamers which is currently only known to reside in the cell membrane [47]. Many of the experimental observations would positively support RUS proteins as transporters of vitamin B6 molecules, though the two most critical are: i) An exogenous additional supply of all of the vitamers is able to at least partially recover the RUS mutant phenotypes [40]. ...
Malaria is a burdensome disease to humanity caused chiefly by the still poorly understood parasite genus Plasmodium . Much of the pathogenic success of these and other related parasites is due to the presence of the apicoplast, a comparatively poorly characterised biosynthetic organelle containing many proteins of unknown function. Here we present AlphaFold2 protein structure predictions together with further in silico analyses to infer molecular functions for the three uncharacterised transmembrane apicoplast proteins PF3D7_0622700, PF3D7_0908100 and PF3D7_1021300. The targets PF3D7_0622700 and PF3D7_0908100 are shown herein to belong to the polytopic Major Facilitator and Cation-Proton Antiporter and Anion Transporter superfamilies respectively, confirming previous suspicions for PF3D7_0908100 of a transporter function. Importantly, our docking screens further suggest pyridoxal-5-phosphate may be transported by PF3D7_0622700, and PF3D7_0908100 likely transports a larger negatively charged metabolite. These findings will help direct experimental assays to confirm what apicoplast metabolites these proteins may transport. PF3D7_1021300 is proposed to possess a six transmembrane alpha-helix domain of a currently unknown fold which may also possess a transporter molecular function. This work highlights the power of high accuracy protein structure predictions to illuminate proteins of unknown structure and function.
... Arabidopsis PUP14 regulates cytokinin signalling output via cytokinin uptake at the plasma membrane (Zürcher et al., 2016). PUP family members have also been shown to function in uptake of a number of non-cytokinin purine compounds (Gillissen et al., 2000;Szydlowski et al., 2013). Overall, a number of PUP transporters have been characterised; however, demonstration of transport specificity and direction has not been universally demonstrated. ...
Development of symbiotic root nodules is a cytokinin-dependent process that is critical to nitrogen acquisition in legumes. The extent and manner in which root nodules contribute to whole-plant cytokinin and nitrogen supply signalling is unknown.
Using a combination of genetic, biochemical and physiological approaches, we characterised the role of cytokinin synthesis, export and perception in coordination of symbiotic nodule development and shoot growth in the legume Lotus japonicus .
LjPup1 encodes a plasma membrane localised cytokinin exporter with isopentenyladenine (iP) and trans -Zeatin (tZ) export capacity. LjPup1 shows a distinct nodule-specific expression pattern with greatest transcript levels detected in mature nodules. Mutants accumulate more isopentenyladenine riboside (iPR) in nodule tissues and demonstrate hallmarks of reduced cytokinin signalling. Despite normal nodule numbers and function, shoot growth is markedly reduced in Ljpup1 mutants, as well as in mutants impaired in tZ biosynthesis.
We found symbiotic root nodules contribute to shoot growth via export of active cytokinins. A cytokinin exporter in the purine permease family thus contributes to long-distance cytokinin homeostasis regulating plant development.
... Analyses using heterologous expressing systems and competition studies revealed that AtPUP1 could transport a variety of CKs including tZ, iP and kinetin (6-furfurylaminopurine), while AtPUP2 mediates the transport of tZ, cZ, iP and the synthetic CKs benzylaminopurine and kinetin (Gillissen et al. 2000, Bürkle et al. 2003). Due to a lack of genetic and physiological evidence, it is only speculated that the PM-localized AtPUP1 (Szydlowski et al. 2013) has a potential role in importing the substrates from the apoplast into the cell and AtPUP2 may function in phloem loading. Moreover, the PM-localized AtPUP14 is required for depleting apoplastic CK pools through transporting bioactive CKs into cells, thus inhibiting the signaling perception through the PM-localized CK sensors (Zürcher et al. 2016). ...
The sustainable production of crops faces increasing challenges from global climate change and human activities, which leads to increasing instances of many abiotic stressors to plants. Among the abiotic stressors, drought, salinity and excessive levels of toxic metals cause reductions in global agricultural productivity and serious health risks for humans. Cytokinins (CKs) are key phytohormones functioning in both the normal development and stress-responses in plants. Here, we summarized the molecular mechanisms on the biosynthesis, metabolism, transport, and signaling transduction pathways of CKs. CKs act as negative regulators of both the root system architecture (RSA) plasticity and root sodium exclusion in response to salt stress. The functions of CKs in mineral-toxicity tolerance and their detoxification in plants are reviewed. Comparative genomic analyses were performed to trace the origin, evolution and diversification of the critical regulatory networks linking CK signaling and abiotic stress. We found that the production of CKs, their derivates, pathways of signal transduction, and drought-response root growth regulation, are evolutionarily conserved in land plants. In addition, the mechanisms of CK-mediated sodium exclusion under salt stress are suggested for further investigations. In summary, we propose that manipulation of CK levels and their signaling pathways are important for plant abiotic stress and are therefore, potential strategies for meeting the increasing demand for global food production under changing climatic conditions.
... To validate the yeast assay screen data for respective total vitamin contents in three rice tissue types from selected accessions with low, intermediate and high vitamin contents, a precise quantification of their soluble constituent vitamers was performed by HPLC against known amounts of external standards Szydlowski et al., 2013). For B 1 vitamers, profiles were obtained for TDP, TMP, and thiamine. ...
Insufficient dietary intake of micronutrients contributes to the onset of deficiencies termed hidden hunger—a global health problem affecting approximately 2 billion people. Vitamin B1 (thiamine) and vitamin B6 (pyridoxine) are essential micronutrients because of their roles as enzymatic cofactors in all organisms. Metabolic engineering attempts to biofortify rice endosperm—a poor source of several micronutrients leading to deficiencies when consumed monotonously—have led to only minimal improvements in vitamin B1 and B6 contents. To determine if rice germplasm could be exploited for biofortification of rice endosperm, we screened 59 genetically diverse accessions under greenhouse conditions for variation in vitamin B1 and vitamin B6 contents across three tissue types (leaves, unpolished and polished grain). Accessions from low, intermediate and high vitamin categories that had similar vitamin levels in two greenhouse experiments were chosen for in-depth vitamer profiling and selected biosynthesis gene expression analyses. Vitamin B1 and B6 contents in polished seeds varied almost 4-fold. Genes encoding select vitamin B1 and B6 biosynthesis de novo enzymes (THIC for vitamin B1, PDX1.3a–c and PDX2 for vitamin B6) were differentially expressed in leaves across accessions contrasting in their respective vitamin contents. These expression levels did not correlate with leaf and unpolished seed vitamin contents, except for THIC expression in leaves that was positively correlated with total vitamin B1 contents in polished seeds. This study expands our knowledge of diversity in micronutrient traits in rice germplasm and provides insights into the expression of genes for vitamin B1 and B6 biosynthesis in rice.
... The resulting supernatant was decanted and used for analysis in two runs, using 10 and 50 mL injection volumes. Separation and detection of B 6 vitamers by HPLC was performed as described previously (Szydlowski et al., 2013). All standards except PNP were purchased from Sigma-Aldrich. ...
Stunted growth in saline conditions is a signature phenotype of the Arabidopsis SALT OVERLY SENSITIVE mutants (sos1-5) affected in pathways regulating the salt stress response. One of the mutants isolated, sos4, encodes a kinase that phosphorylates pyridoxal (PL), a B6 vitamer, forming the important coenzyme pyridoxal 5′-phosphate (PLP). Here, we show that sos4-1 and more recently isolated alleles are deficient in phosphorylated B6 vitamers including PLP. This deficit is concomitant with a lowered PL level. Ionomic profiling of plants under standard laboratory conditions (without salt stress) reveals that sos4 mutants are perturbed in mineral nutrient homeostasis, with a hyperaccumulation of transition metal micronutrients particularly in the root, accounting for stress sensitivity. This is coincident with the accumulation of reactive oxygen species, as well as enhanced lignification and suberization of the endodermis, although the Casparian strip is intact and functional. Further, micrografting shows that SOS4 activity in the shoot is necessary for proper root development. Growth under very low light alleviates the impairments, including salt sensitivity, suggesting that SOS4 is important for developmental processes under moderate light intensities. Our study provides a basis for the integration of SOS4 derived B6 vitamers into plant health and fitness.
... However, the molecular identity of vitB6 transporter protein in mammals has remained elusive [12,30]. Among eukaryotes, the only vitB6 transporters identified so far are the yeast transporters, Tpn1p [31] and Bsu1 [32], and, recently, PUP1 in plant species Arabidopsis (first to be identified in plants) [33]. ...
Vitamin B6 (vitB6) is a generic term that comprises six interconvertible pyridine compounds. These vitB6 compounds (also called vitamers) are pyridoxine (PN), pyridoxamine (PM), pyridoxal (PL) and their 5′-phosphorylated forms pyridoxine 5′-phosphate (PNP), pyridoxamine 5′-phosphate (PMP) and pyridoxal 5′-phosphate (PLP). VitB6 is an essential nutrient for all living organisms, but only microorganisms and plants can carry out de novo synthesis of this vitamin. Other organisms obtain vitB6 from dietary sources and interconvert its different forms according to their needs via a biochemical pathway known as the salvage pathway. PLP is the biologically active form of vitB6 which is important for maintaining the biochemical homeostasis of the body. In the human body, PLP serves as a cofactor for more than 140 enzymatic reactions, mainly associated with synthesis, degradation and interconversion of amino acids and neurotransmitter metabolism. PLP-dependent enzymes are also involved in various physiological processes, including biologically active amine biosynthesis, lipid metabolism, heme synthesis, nucleic acid synthesis, protein and polyamine synthesis and several other metabolic pathways. PLP is an important vitamer for normal brain function since it is required as a coenzyme for the synthesis of several neurotransmitters including D-serine, D-aspartate, L-glutamate, glycine, γ-aminobutyric acid (GABA), serotonin, epinephrine, norepinephrine, histamine and dopamine. Intracellular levels of PLP are tightly regulated and conditions that disrupt this homeostatic regulation can cause disease. In humans, genetic and dietary (intake of high doses of vitB6) conditions leading to increase in PLP levels is known to cause motor and sensory neuropathies. Deficiency of PLP in the cell is also implicated in several diseases, the most notable example of which are the vitB6-dependent epileptic encephalopathies. VitB6-dependent epileptic encephalopathies (B6EEs) are a clinically and genetically heterogeneous group of rare inherited metabolic disorders. These debilitating conditions are characterized by recurrent seizures in the prenatal, neonatal, or postnatal period, which are typically resistant to conventional anticonvulsant treatment but are well-controlled by the administration of PN or PLP. In addition to seizures, children affected with B6EEs may also suffer from developmental and/or intellectual disabilities, along with structural brain abnormalities. Five main types of B6EEs are known to date, these are: PN-dependent epilepsy due to ALDH7A1 (antiquitin) deficiency (PDE-ALDH7A1) (MIM: 266100), hyperprolinemia type 2 (MIM: 239500), PLP-dependent epilepsy due to PNPO deficiency (MIM: 610090), hypophosphatasia (MIM: 241500) and PLPBP deficiency (MIM: 617290). This chapter provides a review of vitB6 and its different vitamers, their absorption and metabolic pathways in the human body, the diverse physiological roles of vitB6, PLP homeostasis and its importance for human health. Finally, the chapter reviews the inherited neurological disorders affecting PLP homeostasis with a special focus on vitB6-dependent epileptic encephalopathies (B6EEs), their different subtypes, the pathophysiological mechanism underlying each type, clinical and biochemical features and current treatment strategies.
... Two plant-specific families include the purine permeases (PUP) and ureide permeases (UPS). In Arabidopsis the 21-member PUP family transports purines, cytokinins and pyridoxine (reviewed in Jelesko 2012;Szydlowski et al. 2013). Three of the eight UPS in Arabidopsis move allantoin, uracil, uric acid and xanthine (Desimone et al. 2002;Schmidt et al. 2004;. ...
... Other plant nucleobase transporters from the NCS2 and UPS, PUP and ENT families locate to the plasma membrane. Plant NCS1 transporters are the only characterized nucleobase transporter with a plastid localization (Witz et al. 2012;Rapp et al. 2016;Nguyen et al. 2020), while members of the NCS2, UPS and PUP families locate to the plasma membrane (Collier and Tegeder 2012;Szydlowski et al. 2013;Niopek-Witz et al. 2014). PgNCS1 functions not only in heterologous systems but in planta as well. ...
The Picea glauca genome contains a locus that encodes for a nucleobase cation symporter 1 (PgNCS1). As a gymnosperm, P. glauca belongs to a key taxonomic position for an ongoing evolution-function analysis of viridiplantae nucleobase cation symporter 1 proteins (NCS1). Here the solute transport and binding properties for PgNCS1 are determined through heterologous expression in Saccharomyces cerevisiae strains deficient in functional NCS1 loci. PgNCS1 displays a broad, yet unique, solute specificity profile –common with other plant NCS1. Yeast containing PgNCS1 transport adenine, guanine, hypoxanthine, xanthine and uracil and are sensitive to growth on 8-azaadenine. Neither cytosine nor 5 flourocytosine are transported by PgNCS1 but along with caffeine and uric acid, act as competitive inhibitors of [3H]-adenine and [3H]-hypoxanthine uptake. This transporter displays high affinity for adenine (Km = 2.67 μM), guanine (Ki = 1.71 μM) and hypoxanthine (Ki = 1.82 μM) but lesser affinity for xanthine (Ki = 5.36 μM). Arabidopsis plants that are deficient in their endogenous NCS1, yet carry PgNCS1, show significant uptake of [3H]-adenine. The results support previous studies and together confirm a broad nucleobase transport and binding pattern for plant NCS1 across the viridiplantae.
... Szydlowski et al. showed that AtPUP1 is also involved in the uptake of pyridoxine (vitamin B6), which can be inhibited, among others, by tZ. They also traced AtPUP1 subcellular localization to the plasma membrane [103]. ...
Cytokinins are a class of phytohormones, signalling molecules specific to plants. They act as regulators of diverse physiological processes in complex signalling pathways. It is necessary for plants to continuously regulate cytokinin distribution among different organs, tissues, cells, and compartments. Such regulatory mechanisms include cytokinin biosynthesis, metabolic conversions and degradation, as well as cytokinin membrane transport. In our review, we aim to provide a thorough picture of the latter. We begin by summarizing cytokinin structures and physicochemical properties. Then, we revise the elementary thermodynamic and kinetic aspects of cytokinin membrane transport. Next, we review which membrane-bound carrier proteins and protein families recognize cytokinins as their substrates. Namely, we discuss the families of “equilibrative nucleoside transporters” and “purine permeases”, which translocate diverse purine-related compounds, and proteins AtPUP14, AtABCG14, AtAZG1, and AtAZG2, which are specific to cytokinins. We also address long-distance cytokinin transport. Putting all these pieces together, we finally discuss cytokinin distribution as a net result of these processes, diverse in their physicochemical nature but acting together to promote plant fitness.
... Plant vitamins are transported in three patterns: between plants and the environment, between different tissues, and between distinct organelles (Figure 3) (Szydlowski et al., 2013;Miyaji et al., 2015;Martinis et al., 2016). Here, we review the transport of several vitamins in plants. ...
... Vitamin B6 supplementation rescues this phenotype, indicating the uptake of vitamin B6 by plants (Titiz et al., 2006). To identify vitamin B6 transporters by complementary assays, Szydlowski et al., (2013) selected a mutant strain of Saccharomyces cerevisiae, that fails to synthesize or absorb vitamin B6. The researchers expressed Arabidopsis purine permeases (PUPs) in the yeast mutant; PUP1 complements the growth phenotype of the yeast mutant. ...
... However, exogenous vitamin B6 can rescue the phenotype of pdx1.3 but not pdx1.3/pup1. That is, PUP1 transports vitamin B6 from the rhizosphere to within the inner ( Figure 3A) (Szydlowski et al., 2013). ...
Vitamins maintain growth and development in humans, animals, and plants. Because plants serve as essential producers of vitamins, increasing the vitamin contents in plants has become a goal of crop breeding worldwide. Here, we begin with a summary of the functions of vitamins. We then review the achievements to date in elucidating the molecular mechanisms underlying how vitamins are synthesized, transported, and regulated in plants. We also stress the exploration of variation in vitamins by the use of forward genetic approaches, such as quantitative trait locus mapping and genome‐wide association studies. Overall, we conclude that exploring the diversity of vitamins could provide new insights into plant metabolism and crop breeding.
... Three genes, AtPUP1, AtPUP2, and AtPUP14, are supposed to mediate CK nucleobase uptake in Arabidopsis (Bürkle et al., 2003;Zürcher et al., 2016). AtPUP1 is expressed in the epithem of hydatodes and the stigma surface of silique, and localized to the plasma membrane, whereas AtPUP2 is expressed in the phloem of leaves (Bürkle et al., 2003;Szydlowski et al., 2013). AtPUP14 is also localized to the plasma membrane, and has the ability to import CK nucleobase into cell (Zürcher et al., 2016). ...
Cytokinins (CKs) are a class of phytohormones playing essential roles in various biological processes. However, the mechanisms underlying CK transport as well as its function in plant growth and development are far from being fully elucidated. Here, we characterize the function of PURINE PERMEASE1 (OsPUP1) in rice (Oryza sativa L.). OsPUP1 was predominantly expressed in the root, particularly in vascular cells, and CK treatment can induce its expression. Subcellular localization analysis showed that OsPUP1 was predominantly localized to the endoplasmic reticulum (ER). Overexpression of OsPUP1 resulted in growth defect of various aerial tissues, including decreased leaf length, plant height, grain weight, panicle length, and grain number. Hormone profiling revealed that the CK content was decreased in the shoot of OsPUP1-overexpressing seedling, but increased in the root, compared with the wild type. The CK content in the panicle was also decreased. Quantitative reverse transcription-PCR (qRT-PCR) analysis using several CK type-A response regulators (OsRRs) as the marker genes suggested that the CK response in the shoot of OsPUP1-overexpressing seedling is decreased compared to the wild type when CKs are applied to the root. Genetic analysis revealed that BG3/OsPUP4, a putative plasma membrane-localized CK transporter, overcomes the function of OsPUP1. We hypothesize that OsPUP1 might be involved in importing CKs into ER to unload CKs from the vascular tissues by cell-to-cell transport.
