Enhancement of vitamin B6 levels in rice expressing Arabidopsis vitamin B6 biosynthesis de novo genes

Vitamin B₆ (pyridoxine) is vital for key metabolic reactions and reported to have antioxidant properties in planta. Therefore, enhancement of vitamin B₆ content has been hypothesized to be a route to improve resistance to biotic and abiotic stresses. Most of the current studies on vitamin B₆ in plants are on eudicot species, with monocots remaining largely unexplored. In this study, we investigated vitamin B₆ biosynthesis in rice, with a view to examining the feasibility and impact of enhancing vitamin B₆ levels. Constitutive expression in rice of two Arabidopsis thaliana genes from the vitamin B₆ biosynthesis de novo pathway, AtPDX1.1 and AtPDX2, resulted in a considerable increase in vitamin B₆ in leaves (up to 28.3‐fold) and roots (up to 12‐fold), with minimal impact on general growth. Rice lines accumulating high levels of vitamin B₆ did not display enhanced tolerance to abiotic stress (salt) or biotic stress (resistance to Xanthomonas oryzae infection). While a significant increase in vitamin B₆ content could also be achieved in rice seeds (up to 3.1‐fold), the increase was largely due to its accumulation in seed coat and embryo tissues, with little enhancement observed in the endosperm. However, seed yield was affected in some vitamin B₆‐enhanced lines. Notably, expression of the transgenes did not affect the expression of the endogenous rice PDX genes. Intriguingly, despite transgene expression in leaves and seeds, the corresponding proteins were only detectable in leaves and could not be observed in seeds, possibly pointing to a mode of regulation in this organ.     

Recycling of pyridoxine (vitamin B6) by PUP1 in Arabidopsis

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 taken up from exogenous sources in a broad range of organisms, including plants. However, specific transporters have been identified only in yeast. Here we assess the ability of the family of Arabidopsis purine permeases (PUPs) to transport vitamin B6. Several members of the family complement the growth phenotype of a Saccharomyces cerevisiae mutant strain impaired in both de novo biosynthesis of vitamin B6 as well as its uptake. The strongest activity was observed with PUP1 and was confirmed by direct measurement of uptake in yeast as well as in planta, defining PUP1 as a high affinity transporter for pyridoxine. At the tissue level the protein is localised to hydathodes and here we use confocal microscopy to illustrate that at the cellular level it is targeted to the plasma membrane. Interestingly, we observe alterations in pyridoxine recycling from the guttation sap upon overexpression of PUP1 and in a pup1 mutant, consistent with the role of the protein in retrieval of pyridoxine. Furthermore, combining the pup1 mutant with a vitamin B6 de novo biosynthesis mutant (pdx1.3) corroborates that PUP1 is involved in the uptake of the vitamin.     

Vitamin B6 deficient plants display increased sensitivity to high light and photo-oxidative stress

Background  

Vitamin B6 is a collective term for a group of six interconvertible compounds: pyridoxine, pyridoxal, pyridoxamine and their phosphorylated derivatives. Vitamin B6 plays essential roles as a cofactor in a range of biochemical reactions. In addition, vitamin B6 is able to quench reactive oxygen species in vitro, and exogenously applied vitamin B6 protects plant cells against cell death induced by singlet oxygen (¹O₂). These results raise the important question as to whether plants employ vitamin B6 as an antioxidant to protect themselves against reactive oxygen species.     

Identification and characterization of a pyridoxal reductase involved in the vitamin B6 salvage pathway in Arabidopsis

Vitamin B6 (pyridoxal phosphate) is an essential cofactor in enzymatic reactions involved in numerous cellular processes and also plays a role in oxidative stress responses. In plants, the pathway for de novo synthesis of pyridoxal phosphate has been well characterized, however only two enzymes, pyridoxal (pyridoxine, pyridoxamine) kinase (SOS4) and pyridoxamine (pyridoxine) 5' phosphate oxidase (PDX3), have been identified in the salvage pathway that interconverts between the six vitamin B6 vitamers. A putative pyridoxal reductase (PLR1) was identified in Arabidopsis based on sequence homology with the protein in yeast. Cloning and expression of the AtPLR1 coding region in a yeast mutant deficient for pyridoxal reductase confirmed that the enzyme catalyzes the NADPH-mediated reduction of pyridoxal to pyridoxine. Two Arabidopsis T-DNA insertion mutant lines with insertions in the promoter sequences of AtPLR1 were established and characterized. Quantitative RT-PCR analysis of the plr1 mutants showed little change in expression of the vitamin B6 de novo pathway genes, but significant increases in expression of the known salvage pathway genes, PDX3 and SOS4. In addition, AtPLR1 was also upregulated in pdx3 and sos4 mutants. Analysis of vitamer levels by HPLC showed that both plr1 mutants had lower levels of total vitamin B6, with significantly decreased levels of pyridoxal, pyridoxal 5'-phosphate, pyridoxamine, and pyridoxamine 5'-phosphate. By contrast, there was no consistent significant change in pyridoxine and pyridoxine 5'-phosphate levels. The plr1 mutants had normal root growth, but were significantly smaller than wild type plants. When assayed for abiotic stress resistance, plr1 mutants did not differ from wild type in their response to chilling and high light, but showed greater inhibition when grown on NaCl or mannitol, suggesting a role in osmotic stress resistance. This is the first report of a pyridoxal reductase in the vitamin B6 salvage pathway in plants.     

Pyridoxine is required for post-embryonic root development and tolerance to osmotic and oxidative stresses

Pyridoxine (vitamin B6) is a cofactor required by numerous enzymes in all cellular organisms. Plants are the major source of vitamin B6 for animals, yet the biosynthesis pathway and the function of vitamin B6 in plants are not well elucidated. In this study, an Arabidopsis pyridoxine synthase gene PDX1 was characterized and its in vivo functions were investigated. The PDX1 gene was expressed in all plant parts examined and its expression level was not significantly regulated by abiotic stress or the phytohormone abscisic acid. In roots, PDX1 was highly expressed in a defined region behind the root tips that undergoes rapid cell division. The PDX1 protein was mainly associated with the plasma membrane and endomembranes, implying a potential involvement of vitamin B6 in membrane function. To reveal the in vivo role of PDX1, Arabidopsis insertional mutants were isolated. Strikingly, the pdx1 knockout mutants were impaired in root growth and early seedling development. The stunted roots resulted from both reduced cell division and elongation. Supplementation of the growth media with pyridoxine or reintroduction of the wild-type PDX1 gene into the mutants completely restored the mutant growth, demonstrating that PDX1 is required for pyridoxine biosynthesis in planta. In addition to the developmental defects, pdx1 mutants are hypersensitive to osmotic stress and oxidative stress. These mutant seedlings had increased peroxidation of membrane lipids following UV treatment. Our study establishes a critical role of vitamin B6 in plant development and stress tolerance and suggests that vitamin B6 may represent a new class of antioxidant in plants.     

Allantoin accumulation through overexpression of ureide permease1 improves rice growth under limited nitrogen conditions

In legumes, nitrogen (N) can be stored as ureide allantoin and transported by ureide permease (UPS) from nodules to leaves where it is catabolized to release ammonium and assimilation to amino acids. In non‐leguminous plants especially rice, information on its roles in N metabolism is scarce. Here, we show that OsUPS1 is localized in plasma membranes and are highly expressed in vascular tissues of rice. We further evaluated an activation tagging rice overexpressing OsUPS1 (OsUPS1^OX) under several N regimes. Under normal field conditions, panicles from OsUPS1^OX plants (14 days after flowering (DAF)) showed significant allantoin accumulation. Under hydroponic system at the vegetative stage, plants were exposed to N‐starvation and measured the ammonium in roots after resupplying with ammonium sulphate. OsUPS1^OX plants displayed higher ammonium uptake in roots compared to wild type (WT). When grown under low‐N soil supplemented with different N‐concentrations, OsUPS1^OX exhibited better growth at 50% N showing higher chlorophyll, tiller number and at least 20% increase in shoot and root biomass relative to WT. To further confirm the effects of regulating the expression of OsUPS1, we evaluated whole‐body‐overexpressing plants driven by the GOS2 promoter (OsUPS1^GOS2) as well as silencing plants (OsUPS1^RNAi). We found significant accumulation of allantoin in leaves, stems and roots of OsUPS1^GOS2 while in OsUPS1^RNAi allantoin was significantly accumulated in roots. We propose that OsUPS1 is responsible for allantoin partitioning in rice and its overexpression can support plant growth through accumulation of allantoin in sink tissues which can be utilized when N is limiting.     

Receptor‐like kinase OsSIK1 improves drought and salt stress tolerance in rice (Oryza sativa) plants

Receptor‐like kinases (RLKs) play essential roles in plant growth, development and responses to environmental stresses. A putative RLK gene, OsSIK1, with extracellular leucine‐rich repeats was cloned and characterized in rice (Oryza sativa). OsSIK1 exhibits kinase activity in the presence of Mn²⁺, and the OsSIK1 kinase domain has the ability to autophosphorylate and phosphorylate myelin basic protein (MBP). OsSIK1 promoter‐GUS analysis revealed that OsSIK1 is expressed mainly in the stem and spikelet in rice. The expression of OsSIK1 is mainly induced by salt, drought and H₂O₂ treatments. Transgenic rice plants with overexpression of OsSIK1 show higher tolerance to salt and drought stresses than control plants. On the contrary, the knock‐out mutants sik1‐1 and sik1‐2, as well as RNA interference (RNAi) plants, are sensitive to drought and salt stresses. The activities of peroxidase, superoxide dismutase and catalase are enhanced significantly in OsSIK1‐overexpressing plants. Also, the accumulation of H₂O₂ in leaves of OsSIK1‐overexpressing plants is much less than that of the mutants, RNAi plants and control plants, as measured by 3,3′‐diamino benzidine (DAB) staining. We also show that OsSIK1 affects stomatal density in the abaxial and adaxial leaf epidermis of rice. These results indicate that OsSIK1 plays important roles in salt and drought stress tolerance in rice, through the activation of the antioxidative system.     

Overexpression of a non-native deoxyxylulose-dependent vitamin B6 pathway in Bacillus subtilis for the production of pyridoxine

Vitamin B6 is a designation for the vitamers pyridoxine, pyridoxal, pyridoxamine, and their respective 5′-phosphates. Pyridoxal 5′-phosphate, the biologically most-important vitamer, serves as a cofactor for many enzymes, mainly active in amino acid metabolism. While microorganisms and plants are capable of synthesizing vitamin B6, other organisms have to ingest it. The vitamer pyridoxine, which is used as a dietary supplement for animals and humans is commercially produced by chemical processes. The development of potentially more cost-effective and more sustainable fermentation processes for pyridoxine production is of interest for the biotech industry. We describe the generation and characterization of a Bacillus subtilis pyridoxine production strain overexpressing five genes of a non-native deoxyxylulose 5′-phosphate-dependent vitamin B6 pathway. The genes, derived from Escherichia coli and Sinorhizobium meliloti, were assembled to two expression cassettes and introduced into the B. subtilis chromosome. in vivo complementation assays revealed that the enzymes of this pathway were functionally expressed and active. The resulting strain produced 14 mg/l pyridoxine in a small-scale production assay. By optimizing the growth conditions and co-feeding of 4-hydroxy-threonine and deoxyxylulose the productivity was increased to 54 mg/l. Although relative protein quantification revealed bottlenecks in the heterologous pathway that remain to be eliminated, the final strain provides a promising basis to further enhance the production of pyridoxine using B. subtilis.     

A rice really interesting new gene H2‐type E3 ligase,OsSIRH2‐14, enhances salinity tolerance via ubiquitin/26S proteasome‐mediated degradation of salt‐related proteins

Salinity is a deleterious abiotic stress factor that affects growth, productivity, and physiology of crop plants. Strategies for improving salinity tolerance in plants are critical for crop breeding programmes. Here, we characterized the rice (Oryza sativa) really interesting new gene (RING) H2‐type E3 ligase, OsSIRH2‐14 (previously named OsRFPH2‐14), which plays a positive role in salinity tolerance by regulating salt‐related proteins including an HKT‐type Na⁺ transporter (OsHKT2;1). OsSIRH2‐14 expression was induced in root and shoot tissues treated with NaCl. The OsSIRH2‐14‐EYFP fusion protein was predominately expressed in the cytoplasm, Golgi, and plasma membrane of rice protoplasts. In vitro pull‐down assays and bimolecular fluorescence complementation assays revealed that OsSIRH2‐14 interacts with salt‐related proteins, including OsHKT2;1. OsSIRH2‐14 E3 ligase regulates OsHKT2;1 via the 26S proteasome system under high NaCl concentrations but not under normal conditions. Compared with wild type plants, OsSIRH2‐14‐overexpressing rice plants showed significantly enhanced salinity tolerance and reduced Na⁺ accumulation in the aerial shoot and root tissues. These results suggest that the OsSIRH2‐14 RING E3 ligase positively regulates the salinity stress response by modulating the stability of salt‐related proteins.     

Functional study of a salt‐inducible TaSR gene in Triticum aestivum

The gene expression chip of a salt‐tolerant wheat mutant under salt stress was used to clone a salt‐induced gene with unknown functions. This gene was designated as TaSR (Triticum aestivum salt‐response gene) and submitted to GenBank under accession number EF580107. Quantitative polymerase chain reaction (PCR) analysis showed that gene expression was induced by salt stress. Arabidopsis and rice (Oryza sativa) plants expressing TaSR presented higher salt tolerance than the controls, whereas AtSR mutant and RNA interference rice plants were more sensitive to salt. Under salt stress, TaSR reduced Na⁺ concentration and improved cellular K⁺ and Ca²⁺ concentrations; this gene was also localized on the cell membrane. β‐Glucuronidase (GUS) staining and GUS fluorescence quantitative determination were conducted through fragmentation cloning of the TaSR promoter. Salt stress‐responsive elements were detected at 588–1074 bp upstream of the start codon. GUS quantitative tests of the full‐length promoter in different tissues indicated that promoter activity was highest in the leaf under salt stress. Bimolecular fluorescence complementation and yeast two‐hybrid screening further showed the correlation of TaSR with TaPRK and TaKPP. In vitro phosphorylation of TaSR and TaPRK2697 showed that TaPRK2697 did not phosphorylate TaSR. This study revealed that the novel TaSR may be used to improve plant tolerance to salt stress.     

A consortium of rhizobacterial strains and biochemical growth elicitors improve cold and drought stress tolerance in rice (Oryza sativa L.)

In the present study, a consortium of two rhizobacteria Bacillus amyloliquefaciens Bk7 and Brevibacillus laterosporus B4, termed ‘BB’, biochemical elicitors salicylic acid and β‐aminobutyric acid (SB) and their mixture (BBSB) were investigated for cold and drought stress tolerance in rice plants. After withholding water for 16 days, rice plants treated with BBSB showed 100% survival, improved seedling height (35.4 cm), shoot number (6.12), and showed minimum symptoms of chlorosis (19%), wilting (4%), necrosis (6%) and rolling of leaves. Similarly, BB inoculation enhanced plant growth and reduced overall symptoms in rice seedlings subjected to 0 ± 5 °C for 24 h. Our results imply several mechanisms underlying BB‐ and BBSB‐elicited stress tolerance. In contrast to the control, both treatments significantly decreased leaf monodehydroascorbate (MDA) content and electrolyte leakage, and increased leaf proline and cholorophyll content. Moreover, activities of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) increased 3.0‐ and 3.6‐fold, respectively. Moreover, expression of OsMYB3R‐2, OsDIL, OsDREB1A and OsCDPK13 genes was significantly up‐regulated, suggesting that these genes play important roles in abiotic stress tolerance of rice. In addition, bacterial strains Bk7 and B4 were able to produce high amounts of IAA and siderophores, and colonise the plant roots, while only strain Bk7 exhibited the capability to form biofilms and solubilise inorganic phosphate. This study indicates that the BB and BBSB bio‐formulations can be used to confer induced systematic tolerance and improve the health of rice plants subject to chilling and drought stress.