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Summary 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., Significance Statement We demonstrate that vitamin B6 can be enhanced in a monocot species (rice), leading to increased accumulation of unphosphorylated B6 vitamers and pyridoxine glucosides, particularly in leaves. Our analysis reveals current limitations and possibly regulatory mechanisms for vitamin B6 biosynthesis in rice endosperm.

Vitamin B₆ is an essential nutrient in the human diet derived primarily from plant sources. While it is well established as a cofactor for numerous metabolic enzymes, more recently, vitamin B₆ has been implicated as a potent antioxidant. The de novo vitamin B₆ biosynthesis pathway in plants has recently been unraveled and involves only two proteins, PDX1 and PDX2. To provide more insight into the effect of the compound on plant development and its role as an antioxidant, we have overexpressed the PDX proteins in Arabidopsis, generating lines with considerably higher levels of the vitamin in comparison with other recent attempts to achieve this goal. Interestingly, it was possible to increase the level of only one of the two catalytically active PDX1 proteins at the protein level, providing insight into the mechanism of vitamin B₆ homeostasis in planta. Vitamin B₆ enhanced lines have considerably larger vegetative and floral organs and although delayed in pre-reproductive development, do not have an altered overall morphology. The vitamin was observed to accumulate in seeds and the enhancement of its levels was correlated with an increase in their size and weight. This phenotype is predominantly a consequence of embryo enlargement as reflected by larger cells. Furthermore, plants that overaccumulate the vitamin have an increased tolerance to oxidative stress providing in vivo evidence for the antioxidant functionality of vitamin B₆. In particular, the plants show an increased resistance to paraquat and photoinhibition, and they attenuate the cell death response observed in the conditional flu mutant.