Adenosine kinase of Arabidopsis. Kinetic properties and gene expression

Abstract

To assess the functional significance of adenosine salvage in plants, the cDNAs and genes encoding two isoforms of adenosine kinase (ADK) were isolated from Arabidopsis. The ADK1- and ADK2-coding sequences are very similar, sharing 92% and 89% amino acid and nucleotide identity, respectively. Each cDNA was overexpressed in Escherichia coli, and the catalytic activity of each isoform was determined. Both ADKs had similar catalytic properties with a K(m) and V(max)/K(m) for adenosine of 0.3 to 0.5 microM and 5.4 to 22 L min(-1) mg(-1) protein, respectively. The K(m) and V(max)/K(m) for the cytokinin riboside N(6)(isopentenyl) adenosine are 3 to 5 microM and 0.021 to 0.14 L min(-1) mg(-1) protein, respectively, suggesting that adenosine is the preferred substrate for both ADK isoforms. In Arabidopsis plants, both ADK genes are expressed constitutively, with the highest steady-state mRNA levels being found in stem and root. ADK1 transcript levels were generally higher than those of ADK2. ADK enzyme activity reflected relative ADK protein levels seen in immunoblots for leaves, flowers, and stems but only poorly so for roots, siliques, and dry seeds. The catalytic properties, tissue accumulation, and expression levels of these ADKs suggest that they play a key metabolic role in the salvage synthesis of adenylates and methyl recycling in Arabidopsis. They may also contribute to cytokinin interconversion.

Figure 1

DNA hybridization analysis of genomic DNA with ADK probes. Arabidopsis genomic DNA (8 μg/lane) was digested with either HindIII (lanes 2, 7, and 12), EcoRI (lanes 3, 8, and 13), EcoRV (lanes 4, 8, and 14), or XbaI (lanes 5, 10, and 15) and the products separated by electrophoresis through a 1% (w/v) agarose gel. The DNA blots were hybridized with a radiolabeled full-length ADK1 cDNA in a hybridization solution containing either 30% (A) or 50% (B) (v/v) formamide or with the ADK2 cDNA in a 50% (v/v) formamide hybridization buffer and washed with 1× SSC at 42°C. The partial ADK1 cDNA (1 ng) was used as a positive control (lanes 1 and 6) and as a test of hybridization specificity of the ADK2 probe (lane 11). Positions of the λHindIII fragments are shown on the left in kb.

Figure 2

Isolation of ADK fusion proteins and ADK antibodies. A, Each ADK cDNA expressed as a His-tagged fusion protein in E. coli and purified by nickel affinity chromatography. Analysis of overexpressed ADK recombinant proteins in E. coli by SDS-PAGE and Coomassie Blue staining. Lane 1, Molecular mass markers in kD from largest to smallest are 97.4, 66.2, 45, 31, 21.5, and 14.4; lane 2, uninduced culture; lanes 3 and 4, induced ADK1 and ADK2, respectively; lanes 5 and 6, purified ADK1 and ADK2, respectively; lane 7, 10 μg leaf crude extract. B, Proteins from a replicate gel shown in A were transferred to PVDF and reacted with ADK antiserum. ADK breakdown products were detected in the ADK overexpressing cultures, and a 38-kD peptide was detected in the leaf tissue. C, The ADK antiserum was titered on slot blots of purified ADK1 and ADK2 containing the indicated amount of each protein.

Figure 3

Kinetic analysis of ADK1 and ADK2. Results are based on the radiochemical assay using purified His-tagged ADKs as outlined in “Materials and Methods.” In all panels ADK1 and ADK2 are represented by ● and ▴, respectively. A, Determination of the optimal ATP:MgCl₂ by varying the ratio from 0/8:1 to 5:1 while maintaining 4 mm ATP. B, ADK activity in the presence of 1 to 8 mm ATP while maintaining the 4:1 ATP:MgCl₂ ratio. C, Ado concentration was varied from 0.22 to 5 μm. D, Using the optimal assay conditions, the concentration of Pi was increased from 0 to 50 mm. Activity is expressed as the percentage of the activity in the absence of added Pi.

Figure 4

Northern analysis of ADK transcript levels in different organs. RNA was extracted and analyzed by northern hybridization using radiolabeled gene-specific probes for ADK1 (A) and ADK2 (B) as described in “Materials and Methods.” Ethidium bromide-stained ribosomal RNA is shown below each panel. Samples were isolated from leaf (lane 1), flower (lane 2), stem (lane 3), and root (lane 4).

Figure 5

Analysis of ADK protein levels in various organs of Arabidopsis. A, Ten micrograms of total protein from crude extracts prepared from leaves of 3- and 6-week-old plants (lanes 1 and 2), flowers of 4- and 6-week-old plants (lanes 3 and 4); roots (lane 5); siliques (lane 6); stems (lane 7); and dry seeds (lane 8) was separated by SDS-PAGE. Each sample was prepared from organs collected from a pool of 10 plants. ADK detected using a fluorescent substrate, quantified, and expressed as a percentage of the amount detected in stems is indicated below each lane. B, The same extracts analyzed in A were desalted and assayed for ADK activity using the radiochemical assay described in “Materials and Methods.” Activity is expressed as a percentage of total ADK activity in stem tissue (18.9 nmol mg⁻¹ min⁻¹).