The PRK/Rubisco shunt strongly influences Arabidopsis seed metabolism and oil accumulation, affecting more than carbon recycling

The carbon efficiency of storage lipid biosynthesis from imported sucrose in green Brassicaceae seeds is proposed to be enhanced by the PRK/Rubisco shunt, in which ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) acts outside the context of the Calvin–Benson–Bassham cycle to recycle CO2 molecules released during fatty acid synthesis. This pathway utilizes metabolites generated by the nonoxidative steps of the pentose phosphate pathway. Photosynthesis provides energy for reactions such as the phosphorylation of ribulose 5-phosphate by phosphoribulokinase (PRK). Here, we show that loss of PRK in Arabidopsis thaliana (Arabidopsis) blocks photoautotrophic growth and is seedling-lethal. However, seeds containing prk embryos develop normally, allowing us to use genetics to assess the importance of the PRK/Rubisco shunt. Compared with nonmutant siblings, prk embryos produce one-third less lipids—a greater reduction than expected from simply blocking the proposed PRK/Rubisco shunt. However, developing prk seeds are also chlorotic and have elevated starch contents compared with their siblings, indicative of secondary effects. Overexpressing PRK did not increase embryo lipid content, but metabolite profiling suggested that Rubisco activity becomes limiting. Overall, our findings show that the PRK/Rubisco shunt is tightly integrated into the carbon metabolism of green Arabidopsis seeds, and that its manipulation affects seed glycolysis, starch metabolism, and photosynthesis.ISSN:1040-4651ISSN:1531-298XISSN:1532-298

Distinct plastid fructose bisphosphate aldolases function in photosynthetic and non-photosynthetic metabolism in Arabidopsis

Plastid metabolism is critical in both photoautotrophic and heterotrophic plant cells. In chloroplasts, fructose-1,6-bisphosphate aldolase (FBA) catalyses the formation of both fructose 1,6-bisphosphate and sedoheptulose 1,7-bisphosphate within the Calvin–Benson cycle. Three Arabidopsis genes, AtFBA1–AtFBA3, encode plastidial isoforms of FBA, but the contribution of each isoform is unknown. Phylogenetic analysis indicates that FBA1 and FBA2 derive from a recently duplicated gene, while FBA3 is a more ancient paralog. fba1 mutants are phenotypically indistinguishable from the wild type, while both fba2 and fba3 have reduced growth. We show that FBA2 is the major isoform in leaves, contributing most of the measurable activity. Partial redundancy with FBA1 allows both single mutants to survive, but combining both mutations is lethal, indicating a block of photoautotrophy. In contrast, FBA3 is expressed predominantly in heterotrophic tissues, especially the leaf and root vasculature, but not in the leaf mesophyll. We show that the loss of FBA3 affects plastidial glycolytic metabolism of the root, potentially limiting the biosynthesis of essential compounds such as amino acids. However, grafting experiments suggest that fba3 is dysfunctional in leaf phloem transport, and we suggest that a block in photoassimilate export from leaves causes the buildup of high carbohydrate concentrations and retarded growth.ISSN:1460-2431ISSN:0022-095

BETA-AMYLASE9 is a plastidial nonenzymatic regulator of leaf starch degradation

b-Amylases (BAMs) are key enzymes of transitory starch degradation in chloroplasts, a process that buffers the availability of photosynthetically fixed carbon over the diel cycle to maintain energy levels and plant growth at night. However, during vascular plant evolution, the BAM gene family diversified, giving rise to isoforms with different compartmentation and bio logical activities. Here, we characterized BETA-AMYLASE 9 (BAM9) of Arabidopsis (Arabidopsis thaliana). Among the BAMs, BAM9 is most closely related to BAM4 but is more widely conserved in plants. BAM9 and BAM4 share features in cluding their plastidial localization and lack of measurable a-1,4-glucan hydrolyzing capacity. BAM4 is a regulator of starch degradation, and bam4 mutants display a starch-excess phenotype. Although bam9 single mutants resemble the wild-type (WT), genetic experiments reveal that the loss of BAM9 markedly enhances the starch-excess phenotypes of mutants al ready impaired in starch degradation. Thus, BAM9 also regulates starch breakdown, but in a different way. Interestingly, BAM9 gene expression is responsive to several environmental changes, while that of BAM4 is not. Furthermore, overexpres sion of BAM9 in the WT reduced leaf starch content, but overexpression in bam4 failed to complement fully that mutant’s starch-excess phenotype, suggesting that BAM9 and BAM4 are not redundant. We propose that BAM9 activates starch degradation, helping to manage carbohydrate availability in response to fluctuations in environmental conditions. As such, BAM9 represents an interesting gene target to explore in crop species.ISSN:0032-0889ISSN:1532-254

Engrailed protects mouse midbrain dopaminergic neurons against mitochondrial complex I insults

International audienceMice heterozygous for homeobox gene Engrailed-1 display progressive loss of mesencephalic dopaminergic (mDA) neurons. We report that exogenous Engrailed-1 and Engrailed-2 (collectively Engrailed) protect mDA neurons from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a mitochondrial complex I toxin used to model PD in animals. Engrailed enhances the translation of nuclear-encoded mRNAs for two key complex I subunits, Ndufs1 and Ndufs3, and increases complex I activity. Accordingly, in vivo protection against MPTP by Engrailed is antagonized by Ndufs1 siRNA. An association between Engrailed and complex I is further confirmed by the reduced expression of Ndufs1 and Ndufs3 in the substantia nigra pars compacta of Engrailed-1 heterozygous mice. Engrailed also confers in vivo protection against 6-hydroxydopamine and α-synuclein-A30P. Finally, the unilateral infusion of Engrailed into the midbrain increases striatal dopamine content resulting in contralateral amphetamine-induced turning. Therefore, Engrailed is both a survival factor for adult mDA neurons and a regulator of their physiological activity