ITPK1 is an InsP6/ADP phosphotransferase that controls phosphate signaling in Arabidopsis

In plants, phosphate (Pi) homeostasis is regulated by the interaction of PHR transcription factors with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates. Here, we combined different methods to obtain a comprehensive picture of how inositol (pyro)phosphate metabolism is regulated by Pi and dependent on the inositol phosphate kinase ITPK1. We found that inositol pyrophosphates are more responsive to Pi than lower inositol phosphates, a response conserved across kingdoms. With CE-ESI-MS we could separate different InsP7 isomers in Arabidopsis and rice, and identify 4/6-InsP7 and a PP-InsP4 isomer hitherto not reported in plants. We found that the inositol pyrophosphates 1/3-InsP7, 5-InsP7 and InsP8 increase severalfold in shoots after Pi resupply and that tissue-specific accumulation of inositol pyrophosphates relies on ITPK1 activities and MRP5-dependent InsP6 compartmentalization. Notably, ITPK1 is critical for Pi-dependent 5-InsP7 and InsP8 synthesis in planta and its activity regulates Pi starvation responses in a PHR-dependent manner. Furthermore, we demonstrate that ITPK1-mediated conversion of InsP6 to 5-InsP7 requires high ATP concentrations and that Arabidopsis ITPK1 has an ADP phosphotransferase activity to dephosphorylate specifically 5-InsP7 under low ATP. Collectively, our study provides deeper insights into Pi-dependent changes in nutritional and energetic states with the synthesis of regulatory inositol pyrophosphates

Influence of alternating temperature preculture on cryopreservation results for potato shoot tips

Cryopreservation is the most suitable long-term storage method for genetic resources of vegetatively maintained crops like potato. In the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) the DMSO droplet method is applied, and so far more than 1000 accessions are cryopreserved with an average regeneration rate of 58%. New experiments with four potato accessions using alternating temperatures (22/8°C day/night temperature, 8 h photoperiod, 7 d) prior to cryopreservation showed improved regeneration. The influence of this preculture on the shoot tips was studied for two wild, frost resistant species Solanum acaule and S. demissum and for two cultivated, frost sensitive potatoes S. tuberosum ‘Désirée’ and ‘King Edward’. Comparison of liquid and solid media after cryopreservation showed improved regeneration on solid media with higher regeneration percentages, less callus formation and better plantlet structure. In comparative analyses biochemical factors like soluble sugars, starch, and amino acid concentrations were measured. Shoot tips after constant and after alternating temperature preculture were analyzed. Total concentrations of soluble sugars (glucose, fructose, and sucrose) were higher for all accessions after the alternating temperature preculture, which could be the reason for improved cryopreservation results

Analysis of subcellular metabolite levels of potato tubers (Solanum tuberosum) displaying alterations in cellular or extracellular sucrose metabolism

The expression of a heterologous invertase in potato tubers (Solanum tuberosum) in either the cytosol or apoplast leads to a decrease in total sucrose content and to an increase in glucose. Depending on the targeting of the enzyme different changes in phenotype and metabolism of the tubers occur: the cytosolic invertase expressing tubers show an increase in the glycolytic flux, accumulation of amino acids and organic acids, and the appearance of novel disaccharides; however, these changes are not observed when the enzyme is expressed in the apoplast [Roessner et al. (2001). Plant Cell, 13, 11-29]. The analysis of these lines raised several questions concerning the regulation of compartmentation of metabolites in potato tubers. In the current study we addressed these questions by performing comparative subcellular metabolite profiling. We demonstrate that: (i) hexoses accumulate in the vacuole independently of their site of production, but that the cytosolic invertase expression led to a strong increase in the cytosolic glucose concentration and decrease in cytosolic sucrose, whereas these effects were more moderate in the apoplastic expressors; (ii) three out of four of the novel compounds found in the cytosolic overexpressors accumulate in the same compartment; (iii) despite changes in absolute cellular content the subcellular distribution of amino acids was invariant in the invertase overexpressing tubers. These results are discussed in the context of current models of the compartmentation of primary metabolism in heterotrophic plant tissues

Downregulation of pyrophosphate: d-fructose-6-phosphate 1-phosphotransferase activity in sugarcane culms enhances sucrose accumulation due to elevated hexose-phosphate levels

Analyses of transgenic sugarcane clones with 45–95% reduced cytosolic pyrophosphate: d-fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) activity displayed no visual phenotypical change, but significant changes were evident in in vivo metabolite levels and fluxes during internode development. In three independent transgenic lines, sucrose concentrations increased between three- and sixfold in immature internodes, compared to the levels in the wildtype control. There was an eightfold increase in the hexose-phosphate:triose-phosphate ratio in immature internodes, a significant restriction in the triose phosphate to hexose phosphate cycle and significant increase in sucrose cycling as monitored by 13C nuclear magnetic resonance. This suggests that an increase in the hexose-phosphate concentrations resulting from a restriction in the conversion of hexose phosphates to triose phosphates drive sucrose synthesis in the young internodes. These effects became less pronounced as the tissue matured. Decreased expression of PFP also resulted in an increase of the ATP/ADP and UTP/UDP ratios, and an increase of the total uridine nucleotide and, at a later stage, the total adenine nucleotide pool, revealing strong interactions between PPi metabolism and general energy metabolism. Finally, decreased PFP leads to a reduction of PPi levels in older internodes indicating that in these developmental stages PFP acts in the gluconeogenic direction. The lowered PPi levels might also contribute to the absence of increases in sucrose contents in the more mature tissues of transgenic sugarcane with reduced PFP activity

Impact of elevated cytosolic and apoplastic invertase activity on carbon metabolism during potato tuber development

During tuberization in Solanum tuberosum var. Desiree maximal catalytic activities of invertase(s) and sucrose synthase are inversely correlated. During the early stages, invertase activity is high and declines during maturation. The decrease in invertase activity is accompanied by a decrease in the hexose to sucrose ratio and an increase in sucrose synthase activity. This switch is paralleled by the onset of the storage phase as shown by the accumulation of starch and storage proteins. Biochemical and genetic evidence suggests that sucrose synthase activity is positively correlated with sink strength. To explore the possibility of enhancing sink strength in potato tubers by elevating the sucrolytic capacity, transgenic potato plants expressing either cytosolic or apoplastic yeast invertase in their tubers were made. Surprisingly, cytosolic invertase led to a decrease and apoplastic invertase to an increase in tuber yield. To understand the causes of the observed phenotypes, carbon metabolism in tubers of transgenic and control plants was analysed during different stages of tuber development. Both cytosolic and apoplastic invertase resulted in decreased sucrose and elevated glucose contents, indicating that sucrose is accessible in both compartments. Metabolic perturbation, however, was found to be compartment specific. Elevated cytosolic invertase activity led to increased carbon flux towards glycolysis and accumulation of phosphorylated intermediates. The phosphorylated intermediates were not used to build up starch, In contrast, apoplastic invertase does not lead to a significant increase in hexose phosphates compared to untransformed controls. Thus, hexoses originating in the apoplast are not efficiently phosphorylated during potato tuber development, which might be explained by an endocytotic uptake of sucrose and/or hexoses from the apoplast into the vacuole bypassing the cytosolic compartment