4, GCI). Barnidipine (Bieliski, 1973). The P concentration in ground solutions is usually high but the assimilable form, inorganic phosphate (Pi), is usually Barnidipine always in a limiting concentration ranging from 1 to 10 catalytic subunit and and regulatory subunits. In Arabidopsis (plants, and genes involved in light signaling, protein degradation, and GTP signaling were down-regulated. All of these results suggest that the AKIN10 catalytic subunit of the SnRK1 complex might be involved in the regulation of some of the metabolic changes that are observed during Pi starvation. RESULTS Barnidipine Expression and Activity of SnRK1 during Phosphate Starvation It has been very well documented that kinases of the AMPK/SnRK1/SNF1 family are activated in low-energy conditions and when Glc is usually absent from your medium (Baena-Gonzlez and Sheen, 2008). In plants, Pi starvation is usually a condition that produces an important reduction in the amount of ATP. To get Rabbit Polyclonal to MN1 information about the regulation of SnRK1 during Pi deficiency, its kinase activity was measured by the phosphorylation of the SAMS peptide. The SnRK1 activity was lower in leaf extracts from Pi-starved plants (Fig. 1). To evaluate if the difference in activity was due to differential expression of the catalytic subunits, we searched the Genevestigator database and found that only two of the three genes that encode the catalytic subunits (and and transcripts. Amplification of was used as an internal control to indicate equal loading. is usually a Pi starvation-inducible gene and was used as an indication of the Pi status (Burleigh and Harrison, 1999). In contrast with and induction was higher in Pi-starved plants and was observed after 5 d of treatment (Fig. 2B). Open in a separate window Physique 1. SnRK1 activity in plants produced for 10 d in Pi-sufficient (+Pi) and Pi-starvation (?Pi) conditions. Activity was estimated using the SAMS peptide kinase assay. Bars symbolize means sd (= 3). Open in a separate window Physique 2. A, Average transmission of gene expression for derived from the microarray data available at the Genevestigator database (Zimmermann et al., 2005) and arranged according to tissue. B, Effect of Pi starvation on the accumulation of and transcripts. RT-PCR of total RNA from rosette leaves from plants produced for 5 and 10 d under Pi-sufficient (+) and Pi-starvation (?) conditions. is usually a Pi-starvation control gene, and was used as an internal control. [Observe online article for color version of this physique.] Phosphate Starvation Induces Differential Changes in AKIN10 and AKIN11 Catalytic Subunits Having established that no changes in gene expression were observed in the catalytic subunits during Pi starvation to explain the reduction in activity, we analyzed the protein levels. Transgenic Arabidopsis plants transporting AKIN10-GFP and AKIN11-GFP fusion proteins were evaluated. Localization of GFP fusion proteins by confocal microscopy showed that both catalytic subunits colocalized with the chlorophyll, suggesting a chloroplast localization (Fig. 3, A and D). In the case of AKIN10, a low but detectable transmission was also observed in the cytoplasm (Fig. 3C). To confirm the localization of the catalytic subunits, we raised polyclonal antibodies using a polypeptide that acknowledged AKIN10 and AKIN11 to detect the subunits in root and leaf sections. Immunolocalization analysis indicated that this catalytic Barnidipine subunits are present in both organs (Fig. 4, A and G). In leaf cells, the catalytic subunits are localized in the chloroplast but the transmission was also detected in the cytoplasm (Fig. 4, ACC). In roots, the catalytic subunits were present in all cells, from the epidermis to the vascular tissue (Fig. 4, GCI). To probe the chloroplast localization of the catalytic subunits at the biochemical level, proteins obtained from purified chloroplasts were separated with SDS-PAGE and transferred to a nitrocellulose membrane. Western-blot analysis recognized a polypeptide band around 58 kD, which is the expected molecular mass for AKIN11 and AKIN10. The same blot was probed with Rubisco antibodies (Fig. 5). Under Pi starvation conditions, the detection of GFP fusion proteins showed no important changes in AKIN10. Interestingly, the fluorescence corresponding to AKIN11-GFP almost completely disappeared (Fig. 6). This reduction in the fluorescence corresponding to one of the catalytic subunits was also observed in the leaf and root sections of Pi-starved plants, visualized using the anti-= 3). B, Protein extracts from plants overexpressing AKIN10-GFP were separated by SDS-PAGE and transferred to nitrocellulose membranes. Immunodetection was carried out using anti-GFP antibodies. C, Protein extracts from plants overexpressing AKIN11-GFP were separated and immunodetected as explained above. D, Protein extracts from transgenic plants overexpressing AKIN10-GFP were separated and immunodetected using a specific P-Thr antibody. Silencing of Affects Starch Metabolism during Pi Starvation The results presented so far suggest that SnRK1 complexes are created with the AKIN10 catalytic subunit under Pi starvation. To better understand the role of AKIN10 during Pi starvation, a T-DNA insertion collection in Barnidipine was recognized and characterized..