BnaPPT1 is essential for chloroplast development and seed oil accumulation in Brassica napus.

INTRODUCTION: Phosphoenolpyruvate/phosphate translocator (PPT) transports phosphoenolpyruvate from the cytosol into the plastid for fatty acid (FA) and other metabolites biosynthesis. OBJECTIVES: This study investigated PPTs' functions in plant growth and seed oil biosynthesis in oilseed crop Brassica napus. METHODS: We created over-expression and mutant material of BnaPPT1. The plant development, oil content, lipids, metabolites and ultrastructure of seeds were compared to evaluate the gene function. RESULTS: The plastid membrane localized BnaPPT1 was found to be required for normal growth of B. napus. The plants grew slower with yellowish leaves in BnaA08.PPT1 and BnaC08.PPT1 double mutant plants. The results of chloroplast ultrastructural observation and lipid analysis show that BnaPPT1 plays an essential role in membrane lipid synthesis and chloroplast development in leaves, thereby affecting photosynthesis. Moreover, the analysis of primary metabolites and lipids in developing seeds showed that BnaPPT1 could impact seed glycolytic metabolism and lipid level. Knockout of BnaA08.PPT1 and BnaC08.PPT1 resulted in decreasing of the seed oil content by 2.2 to 9.1%, while overexpression of BnaC08.PPT1 significantly promoted the seed oil content by 2.1 to 3.3%. CONCLUSION: Our results suggest that BnaPPT1 is necessary for plant chloroplast development, and it plays an important role in maintaining plant growth and promoting seed oil accumulation in B. napus.

Detection and quantitation of phosphorus metabolites in crude tissue extracts by 1H and 31P NMR: use of gradient assisted 1H-31P HMQC experiments, with selective pulses, for the assignment of less abundant metabolites.

The analysis of crude tissue extracts by NMR has proven to be of use in the study of metabolism due to the non-destructive and non-selective character of the technique. Lists of 1H and 31P NMR assignments of phosphorus metabolites in water solution at specified pH and ionic composition are of large general value but their usefulness may be limited when analysing complex mixtures of metabolites at low concentrations. In this work we report on the use of gradient-assisted proton detected multiple quantum 1H and 31P coherence experiments with selective pulses for the rapid and unambiguous assignments of some crowded regions in 1H and 31P spectra of crude extracts from rat liver. The amplitudes of the gradient episodes were calibrated to optimize the coherence transfer pathway between proton and phosphorus, and the delay for the evolution of the long-range coupling was calculated from values of 3JPH and 4JPH ranging from 1.4 to 7.5 Hz. Moreover, a selective 90 degrees Gaussian pulse on the 31P channel was introduced to increase the resolution in the F1-domain and make the method even faster. The procedure was then applied to unambiguously assign the ID 31P and 1H spectra of perchloric acid extracts of rat livers that had been stimulated with phenylephrine, dBcAMP and glucagon and thus detect changes in the concentration of less abundant metabolites such as phosphoenolpyruvate, UDP-glucose and AMP. The fact that the quantification of these metabolites by either 31P and 1H methods lead to different results is discussed, and the use of 1H NMR spectroscopy for the quantification of phosphorus metabolites whose signal are too weak or poorly resolved in a 31P spectrum is proposed.

Determination of picomole amounts of glycerate 3-phosphate, glycerate 2-phosphate, and phosphoenol pyruvate by an enzymatic assay coupled to firefly luciferase/luciferin luminescence.

A procedure for the determination of picomole amounts of glycerate 3-phosphate, glycerate 2-phosphate, and phosphoenol pyruvate is described. These metabolites were utilized by the glycolytic enzymes phosphoglycerate mutase, enolase, and pyruvate kinase to generate ATP which was determined by firefly luciferase/luciferin luminescence. The phosphoglycerate mutase used was of the glycerate 2,3-bisphosphate-independent type and was prepared from wheat germ. Stoichiometric conversion of glycerate 3-P, ranging in amount from 9 to 275 pmol, occurred after 25 min preincubation and required a narrow range of added mutase. The application of the procedure for determining these metabolites in suspensions of plant protoplasts is described.

The effects of vitamin D on the rat kidney metabolism under conditions of experimental hypercalcemia.

The effects of vitamin D3 on rat kideney metabolism under conditions of experimental hypercalcemia. Acta Physiol. Pol., 1978, 29 (2) 153--159. The effect of vitamin D3 on renal gluconeogenesis processes was studied in the rat. The performed estimations of gluconeogenesis rate from malate and lactate demonstrated significant increases of glucose formation rate when kidney cortex slices of the vitamin D3 treated animals were analyzed. Further studies on the mechanism of the observed phenomenon were performed using kidney cell fractionation procedure and fluorometric estimation of the concentration of selected gluconeogenetic metabolites. Significant increases of phosphoenolpyruvate concentration in the cytosol fraction and citrate concentration in the mitochondrial fraction were observed. Comparison of the described action of vitamin D3 on gluconeogenesis and the effect of parathyriod hormone, as known from the literature suggests similar mechanism of both factor actions. A possibility of vitamin D3 action through the increase in intracellular calcium has been discussed.