Article Figures & Data
Figures
- Figure 1.
Comparison of NMP kinases. A, Comparison of all Arabidopsis and rice NMP kinase proteins containing Inter-Pro signature IPR011769. Predictions of intracellular localization of mature proteins were performed with iPSORT, TargetP, and Predotar. Method intern scores for the predictions are shown, and the results are classified as follows: 0, predicted as having no signal, mitochondrial targeting, or chloroplast transit peptide; M, predicted as having a mitochondrial targeting peptide; P, predicted as having a chloroplast targeting peptide. Our GFP results and the summary of other studies are classified as follows: C, protein found in the cytosol; M, protein associated to mitochondria; P, protein associated to plastids. 1, Millar et al. (2001); 2, Heazlewood et al. (2004); 3, Peltier et al. (2006); 4, Peltier et al. (2004); 5, Carrari et al. (2005); 6, Regierer et al. (2002); *, annotated Os08g0288200 is presumably not full length; therefore, an assembly of CT842017 and AK070372 was translated and used for analysis. B, Fluorescence signals of leaf mesophyll cells. 1, AMK3-GFP, GFP fluorescence signal (green); 2, AMK3-GFP, overlay of GFP and chlorophyll autofluorescence signal (red); 3, AMK2-GFP, GFP fluorescence signal; 4, AMK2-GFP, overlay of GFP and chlorophyll autofluorescence signal; 5, AMK5-GFP, GFP fluorescence signal; 6, AMK5-GFP, overlay of GFP and chlorophyll autofluorescence signal; 7, AMK1-GFP, GFP fluorescence signal; 8, AMK1-GFP, DsRed fluorescence signal of the transiently transformed mitochondrial marker DHODH-DsRed (blue); 9, AMK1-GFP, overlay of GFP, DsRed, and chlorophyll autofluorescence signal; 10, AMK7-GFP, GFP fluorescence signal of transiently transformed protoplasts; 11, AMK7-GFP, overlay of GFP and chlorophyll autofluorescence signal; white bars = 4 μm.
- Figure 2.
Expression and co-response analysis. A, Relative transcript levels of AMK genes. Rosette leaves of 21-d-old plants on soil, separate roots, or complete 16-d-old seedlings on plates containing one-half-strength MS with 0.5% Suc were analyzed by quantitative real-time RT-PCR. Values are expressed as difference of the ct value to EF1α taken to the power of efficiency. Each bar represents the mean values ± sd of three individual seedlings; measurements were repeated twice. B, Co-response analysis of AMK genes. The Arabidopsis co-response database was searched for transcriptional correlations using the nonparametric Spearman's Rho rank correlation and Bonferroni correction with the complete AtGenExpress developmental series in wild-type plants (http://csbdb.mpimp-golm.mpg.de; atge0100: developmental series [only WT]). Visualization was done with MapMan (http://gabi.rzpd.de/projects/MapMan) together with PageMan (http://mapman.mpimp-golm.mpg.de/pageman) and statistical analysis of Wilcoxon's test, including the correction of multiple testing by Bonferroni. P values were assigned a positive or negative value, depending on whether the correlation was positive or negative, with increasing blue and red indicating an increasingly and decreasingly significant correlation, respectively. All significant MapMan categories of metabolic processes that match our high stringent selection are shown.
- Figure 3.
Phenotype of amk2 mutant seeds. A, Structure of Arabidopsis AMK2 and AMK5. Gray lines indicate 5′-untranslated regions, black boxes indicate exons, and black lines are introns. Exons with high similarity are connected with dotted lines. White triangles indicate the position of the T-DNA insertion sites of amk2-1 (Salk_031816), amk2-2 (Gabi-Kat_034C05), and amk5 (Salk_000200). Salk_000200 contains two inserted T-DNAs, 129 bp apart from each other. B, Seeds and embryos. 1, AMK2-1/amk2-1 silique 10 d after flowering (DAF); 2, AMK2-1/amk2-1 silique 14 DAF; 3, AMK2-1/amk2-1 dissected embryos; 4, AMK2-2/amk2-2 dissected embryos; 5 and 6, F2 embryos in late torpedo state from AMK2-2/amk2-2 × AMK2-1/amk2-1; 7 and 8, F2 embryos in early torpedo state from AMK2-2/amk2-2 × AMK2-1/amk2-1. C, Genotype of dissected embryos (14 DAF) of an AMK2-1/amk2-1 plant; lanes 1 to 4, white seeds, lanes 5 to 15, green seeds.
- Figure 4.
Phenotype of amk mutant plants. A, Growth phenotype of amk2 mutant plants; 1, amk2-1/amk2-1; 2, amk2-2/amk2-2; 3, AMK2-2/amk2-2. Plants were grown for 21 d on plates containing one-half-strength MS with 0.5% Suc. Pictures were taken with the same magnification. B, Growth phenotype of amk5 mutant plants; 1, amk5/amk5; 2, AMK5/amk5. Plants were grown for 4 weeks on soil. Pictures were taken with the same magnification.
- Figure 5.
Relative transcript levels of nucleotide metabolism genes in amk mutants. Sixteen-day-old seedlings grown on plates containing one-half-strength MS with 0.5% Suc were analyzed by quantitative real-time RT-PCR. Values are calculated as the difference of the ct value to UBQ-10 taken to the power of efficiency. Each bar represents the expression of the gene of question in the respective mutant to the expression of the wild type. Each bar represents the mean values ± se of three to five individual seedlings. Measurements were repeated twice. The gray bar shades differences that are less than 2-fold. PurA, Adenylosuccinate synthetase EC 6.3.4.4; PurB; adenylosuccinate lyase EC 4.3.2.2; PurC; SAICAR synthase EC 6.3.2.6; PurD, GAR synthase EC 6.3.4.13; PurE, AIR carboxylase and N5CAIR mutase EC 4.1.1.21; PurF, PRPP amidotransferase EC 2.4.2.14; PurH, ACAR transforylase EC 2.1.2.3 and IMP cyclohydrolase EC 3.5.4.10; PurL, FGAM synthase EC 6.3.5.3; PurM, AIR synthase EC 6.3.3.1; PurN, GAR transformylase EC 2.1.2.2; GuaA, GMP synthase EC 6.3.5.2; GuaB, IMP dehydrogenase EC 1.1.1.205; UMK, UMP kinase EC 2.7.4.4; AMK, AMP kinase EC 2.7.4.3; GMK, GMP kinase EC 2.7.4.8; NDK, nucleoside diphosphate kinase EC 2.7.4.6; HGPT, hypoxanthine guanine phosphoribosyltransferase EC 2.4.2.8; APT, adenine phosphoribosyl transferase EC 2.4.2.7; ADK, adenosine kinase EC 2.7.1.20; PRS, PRPP synthetase EC 2.7.6.1. nd, Not detectable.
- Figure 6.
AMK2 specific protein levels in amk mutants. Immunoblot analysis of rosette leaves of 16-d-old seedlings grown on plates containing one-half-strength MS with 0.5% Suc. Size-fractionated proteins were immunodecorated with specific antiserum raised against AMK2. Congenic wild types (lanes 1 and 4), amk2 mutant (lane 2), and amk5 mutant (lane 3) are shown.
- Figure 7.
Analysis of recombinant AMK2 and AMK5 in E. coli lysates. A, Size-fractionated proteins of crude E. coli lysates 5 h after induction of the respective recombinant protein. Lanes 1 to 4, Coomassie staining of total proteins; lanes 5 to 7, immunoblot analysis with His-tag-specific antibody. Lane 1, Marker proteins; lanes 2 and 5, AMK2; lanes 3 and 6, AMK5; lanes 4 and 7, control construct. Arrows indicate the AMK specific proteins of crude E. coli lysates. B, Adenylate kinase activity in crude E. coli lysates 5 h after induction. Total soluble protein extracts were measured in direction of ADP production (backward reaction). Data are the means and sd of three technical replicates of one representative induction.
- Figure 8.
Photosynthesis-related analysis of amk2 mutants. A western blot of rosette leaves of 16-d-old seedlings grown on plates containing one-half-strength MS with 0.5% Suc for ATP-synthase subunits. Size-fractionated proteins were immunodecorated with specific antiserum raised against the ATP-synthase subunit proteins as indicated. Lanes 1 and 8, wild types; lane 2, AMK2-1/amk2-1; lanes 3 and 4, amk2-1/amk2-1; lanes 5 and 6, amk2-2/amk2-2; lane 7, AMK2-2/amk2-2. B, Ultrastructure of chloroplasts from wild-type and amk2 mutant plants. Electron micrographs of leaf tissues. 1, Wild type; 2, homozygous amk2-1; 3 and 4, two individual homozygous amk2-2 plants. All pictures were taken with 16,000× magnification. The leaves were collected from 21-d-old plants grown on one-half-strength MS with 0.5% Suc.
Tables
- Table I.
Activity and nucleotide content in amk mutants
Rosette leaves of seedlings were analyzed after 21 d of growth on plates containing one-half-strength MS with 0.5% Suc. Activity data are given as percentage of the controls, with forward reaction at 4.2 ± 1.3 nmol s−1 (mg protein)−1 and backward reaction at 4.0 ± 1.5 nmol s−1 (mg protein)−1. Each data point represents the mean and sd of seven replicates for amk2, nine replicates for congenic wild-type and heterozygous plants, and four replicates for amk5. Significant differences (P < 0.1) using unpaired two-tailed t tests are marked with an asterisk.
amk2 AMK amk5 Activity Forward reaction (% of control) 79 ± 15.7* 100 ± 19.2 99 ± 7.3 Backward reaction (% of control) 63 ± 14.9* 100 ± 4.3 101 ± 15.5 Metabolite content AMP (nmol g FW−1) 3.9 ± 1.8 5.4 ± 2.4 7.5 ± 1.0 ADP (nmol g FW−1) 8.5 ± 4.5* 25.5 ± 8.8 31.6 ± 2.0 ATP (nmol g FW−1) 18.7 ± 7.4* 50.4 ± 19.4 58.0 ± 10.1 Purine (nmol g FW−1) 32.8 ± 8.4* 89.5 ± 26.2 110 ± 15.9 Adenylate energy charge ([ATP] + [0.5 × ADP]) × ([ATP] + [ADP] + [AMP])−1 0.72 ± 0.09* 0.79 ± 0.05 0.77 ± 0.03 - Table II.
Photosynthesis parameter in amk mutants
Rosette leaves of seedlings were analyzed after 21 d of growth on plates containing one-half-strength MS with 0.5% Suc. ΦPSII and qN were determined using imaging PAM. The values were determined for three plants of the same genotype 5 min after the light (250 μmol photon m−2 s−1) was switched on. Significant differences (P < 0.1) using unpaired two-tailed t tests are marked with an asterisk.
amk2 AMK Chlorophyll content (μg g FW−1) 13.6 ± 5.0* 412 ± 83.9 ΦPSII 0.11 ± 0.011* 0.53 ± 0.040 qN 0.92 ± 0.018* 0.29 ± 0.018
Supplemental Data
Supplemental Table and Figures
Files in this Data Supplement:
- Supplemental Data - Supplemental Table
- Supplemental Data - Supplemental Figure 1
- Supplemental Data - Supplemental Figure 2
