Teresa B. Fitzpatrick

Metabolic effectors are critical in orchestrating biological processes. B vitamin-derived coenzymes are vital for coordinating metabolic enzyme activities, the disruption of which has been linked to numerous diseases in humans and fitness in plants. Modified derivatives of these coenzymes exist and could act as effectors to regulate certain metabolic processes but remain unexplored. Here, we demonstrate that the non-coenzyme vitamin B1 derivative thiamine triphosphate is transiently produced early during the light period in Arabidopsis thaliana (Arabidopsis). The production of this compound is driven by a chemiosmotic mechanism in the chloroplast, which contrasts with mammalian neuronal cells, where it is produced in the mitochondria. Using biophysical techniques of photosynthesis, we show that thiamine triphosphate serves to brake the activation state of chloroplast ATP synthase. This effect is suggested to be achieved by the opportune biosynthesis of thiamine triphosphate from inorganic phosphate and the coenzyme thiamine diphosphate in chloroplasts, which alters the substrate budget of photosynthesis, affecting the coordination of ATP synthesis and consumption during the Calvin-Benson cycle. Intriguingly, the absence of thiamine triphosphate alters the expression of plastid-encoded ATP synthase subunits. The study expands our knowledge on coenzyme derivatives as regulatory molecules in biological systems and, in this case, their regulation of energy metabolism.

Paraquat (PQ) is a fast-acting non-selective herbicide widely used globally to eradicate weeds. The emergence of weed resistance has fueled the drive to understand molecular mechanistic aspects and develop crops resistant to the herbicide. The transport of PQ is mediated by members of the L-amino acid transporter family, which are prime targets for the development of resistance. However, these transporters also facilitate the transport of natural essential molecules such as polyamines and thiamine (vitamin B1), at least in Arabidopsis, but have not undergone rigorous investigation in crops. Here, we report on the disruption of the polyamine transporter PUT3 in two japonica rice cultivars. Both rice put3-mutant alleles are resistant to PQ and display low percentage germination concomitant with altered polyamine profiles, whereas thiamine is unchanged. Notwithstanding, seedlings that germinate behave like wildtype in the Tainung 67 cultivar, whereas further growth and development is strongly impaired by the disruption of PUT3 in the Hwayoung cultivar. The growth phenotype could be complemented by ectopic expression of PUT3, which also restores the polyamine profile thus linking the defects to disruption of the gene. Our study provides biological insight into the divergent characteristics of rice cultivar tissues as a function of their polyamine profile and a warning to exercise caution upon disruption of transporters to facilitate PQ resistance in crops as this may also lead to severe fitness penalties.

B vitamins are a source of coenzymes for a vast array of enzyme reactions, particularly those of metabolism. As metabolism is the basis of decisions that drive maintenance, growth, and development, B vitamin–derived coenzymes are key components that facilitate these processes. For over a century, we have known about these essential compounds and have elucidated their pathways of biosynthesis, repair, salvage, and degradation in numerous organisms. Only now are we beginning to understand their importance for regulatory processes, which are becoming an important topic in plants. Here, I highlight and discuss emerging evidence on how B vitamins are integrated into vital processes, from energy generation and nutrition to gene expression, and thereby contribute to the coordination of growth and developmental programs, particularly those that concern maintenance of a stable state, which is the foundational tenet of plant homeostasis.

Coenzyme management is important for homeostasis of the pool of active metabolic enzymes. The coenzyme pyridoxal 5′-phosphate (PLP) is involved in diverse enzyme reactions including amino acid and hormone metabolism. Regulatory proteins that contribute to PLP homeostasis remain to be explored in plants. Here, we demonstrate the importance of proteins annotated as PLP homeostasis proteins (PLPHPs) for controlling PLP in Arabidopsis (Arabidopsis thaliana). A systematic analysis indicates that while most organisms across kingdoms have a single PLPHP homolog, Angiosperms have two. PLPHPs from Arabidopsis bind PLP and exist as monomers, in contrast to reported PLP-dependent enzymes, which exist as multimers. Disrupting the function of both PLPHP homologs perturbs vitamin B6 (pyridoxine) content, inducing a PLP deficit accompanied by light hypersensitive root growth, unlike PLP biosynthesis mutants. Micrografting studies show that the PLP deficit can be relieved distally between shoots and roots. Chemical treatments probing PLP-dependent reactions, notably those for auxin and ethylene, provide evidence that PLPHPs function in the dynamic management of PLP. Assays in vitro show that Arabidopsis PLPHP can coordinate PLP transfer and withdrawal from other enzymes. This study thus expands our knowledge of vitamin B6 biology and highlights the importance of PLP coenzyme homeostasis in plants.

Coenzyme management is believed to be important for the required pool of active enzymes driving metabolic routes to facilitate homeostasis and match environmental circumstance. The coenzyme pyridoxal 5’-phosphate (PLP) (a vitamin B ₆ derivative) is involved in a diverse array of enzyme reactions spanning amino acid to hormone metabolism. However, dedicated proteins that contribute to PLP homeostasis have not yet been studied in plants. Here we demonstrate the importance of proteins annotated PLP HOMEOSTASIS PROTEINs (PLPHPs) for control of PLP in Arabidopsis. A systematic analysis indicates that while most kingdoms have a single PLPHP homolog, Angiosperms within the plant kingdom have two. PLPHPs from Arabidopsis bind PLP and exist as monomers in solution in contrast to reported PLP-dependent enzymes from all kingdoms. Disrupting functionality of both homologs perturbs vitamin B ₆ content including a PLP deficit accompanied by impaired and light hypersensitive root growth, unlike biosynthesis mutants. Micrografting studies show that the PLP deficit can be relieved distally between shoots and roots. Yet, supplementation experiments do not restore vitamin B ₆ homeostasis in the absence of PLPHP. A series of chemical treatments probing PLP-dependent reactions, notably those for auxin and ethylene, provide evidence that the physiological role of PLPHPs is dynamic management of PLP. Assays in vitro show that Arabidopsis PLPHP can coordinate both PLP transfer and withdrawal. This study expands our broader knowledge of vitamin B ₆ biology and highlights the importance of PLP coenzyme homeostasis in plants, providing a platform for further investigations in boosting adaptive responses. One sentence summary: PLPHPs contribute to surveillance of vitamin B ₆ homeostasis, likely acting as a rheostat in adaptive responses as a function of the use of the coenzyme PLP.

The identification of factors that regulate C/N utilization in plants can make a substantial contribution to optimization of plant health. Here, we explored the contribution of pyridox(am)ine 5′-phosphate oxidase3 (PDX3), which regulates vitamin B₆ homeostasis, in Arabidopsis (Arabidopsis thaliana). Firstly, N fertilization regimes showed that ammonium application rescues the leaf morphological phenotype of pdx3 mutant lines but masks the metabolite perturbance resulting from impairment in utilizing soil nitrate as a source of N. Without fertilization, pdx3 lines suffered a C/N imbalance and accumulated nitrogenous compounds. Surprisingly, exploration of photorespiration as a source of endogenous N driving this metabolic imbalance, by incubation under high CO₂, further exacerbated the pdx3 growth phenotype. Interestingly, the amino acid serine, critical for growth and N management, alleviated the growth phenotype of pdx3 plants under high CO₂, likely due to the requirement of pyridoxal 5′-phosphate for the phosphorylated pathway of serine biosynthesis under this condition. Triggering of thermomorphogenesis by growth of plants at 28 °C (instead of 22 °C) did not appear to require PDX3 function, and we observed that the consequent drive toward C metabolism counters the C/N imbalance in pdx3. Further, pdx3 lines suffered a salicylic acid-induced defense response, probing of which unraveled that it is a protective strategy mediated by nonexpressor of pathogenesis related1 (NPR1) and improves fitness. Overall, the study demonstrates the importance of vitamin B₆ homeostasis as managed by the salvage pathway enzyme PDX3 to growth in diverse environments with varying nutrient availability and insight into how plants reprogram their metabolism under such conditions.

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 B ₆ homeostasis, influences C/N balance. The B ₆ vitamer 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 CO ₂ levels further exacerbates the pdx3 phenotypes. Interestingly, serine supplementation rescues growth under high CO ₂ likely 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 in pdx3 lines that compromises growth, a basal level of SA protects against loss of PDX3 biochemical function. Overall, the study highlights environmental scenarios where vitamin B ₆ homeostasis, as managed by the salvage pathway enzyme PDX3, is critical and provides insight into how plants reprogram their metabolism under such conditions.