Constitutively high expression of the histidine biosynthetic pathway contributes to nickel tolerance in hyperaccumulator plants

Abstract

Plants that hyperaccumulate Ni exhibit an exceptional degree of Ni tolerance and the ability to translocate Ni in large amounts from root to shoot. In hyperaccumulator plants in the genus Alyssum, free His is an important Ni binding ligand that increases in the xylem proportionately to root Ni uptake. To determine the molecular basis of the His response and its contribution to Ni tolerance, transcripts representing seven of the eight enzymes involved in His biosynthesis were investigated in the hyperaccumulator species Alyssum lesbiacum by RNA gel blot analysis. None of the transcripts changed in abundance in either root or shoot tissue when plants were exposed to Ni, but transcript levels were constitutively higher in A. lesbiacum than in the congeneric nonaccumulator A. montanum, especially for the first enzyme in the biosynthetic pathway, ATP-phosphoribosyltransferase (ATP-PRT). Comparison with the weak hyperaccumulator A. serpyllifolium revealed a close correlation between Ni tolerance, root His concentration, and ATP-PRT transcript abundance. Overexpression of an A. lesbiacum ATP-PRT cDNA in transgenic Arabidopsis thaliana increased the pool of free His up to 15-fold in shoot tissue, without affecting the concentration of any other amino acid. His-overproducing lines also displayed elevated tolerance to Ni but did not exhibit increased Ni concentrations in either xylem sap or shoot tissue, suggesting that additional factors are necessary to recapitulate the complete hyperaccumulator phenotype. These results suggest that ATP-PRT expression plays a major role in regulating the pool of free His and contributes to the exceptional Ni tolerance of hyperaccumulator Alyssum species.

Figure 1.

Pathway of His Biosynthesis in Plants. Abbreviations used for enzyme names in this work are shown in parentheses. 2-OG, 2-oxoglutarate. Modified after Ward and Ohta (1999) and Ohta et al. (2000).

Figure 2.

Transcript Abundance of His Biosynthetic Genes in Root Tissue of A. lesbiacum Is Unaffected by Ni. Five-week-old hydroponically grown plants, cultivated in standard nutrient solution not containing Ni, were exposed at the start of the experiment to either 300 μM NiSO₄ (Ni) or an additional 300 μM MgSO₄ (Mg). Four plants were harvested for analysis at various times between 0 and 48 h after exposure to Ni or Mg. Tissue was pooled and total RNA extracted for RNA gel blot analysis using ³²P-labeled partial cDNA probes for each His gene amplified from A. lesbiacum. Expression of β-tubulin transcript (using an A. lesbiacum cDNA probe) was used as a control for equal loading of RNA. Time points of sampling are shown, together with horizontal bar indicating the 16-h-light (white bar)/8-h-dark (black bar) cycle in the growth chamber. The results shown are for one experiment representative of four.

Figure 3.

Nickel Tolerance of Three Species of Alyssum. Plants were cultivated for 6 weeks in standard hydroponic medium supplemented with various concentrations of NiSO₄ before harvesting for dry biomass determination of shoots and roots. Values are means ± se (n = 8 plants). Absolute total plant biomass for control plants grown in the absence of Ni were as follows: A. lesbiacum, 104.0 ± 9.7 mg; A. serpyllifolium, 53.7 ± 2.5 mg; A. montanum, 108.9 ± 7.7 mg.

Figure 4.

RNA Gel Blot Analysis of His Gene Expression in A. lesbiacum and A. montanum. Total RNA was extracted from three independent groups of pooled tissue (four 6-week-old plants per group) from plants grown hydroponically at different times without added Ni. Gene-specific ³²P-labeled 3′-UTR probes for the two Alyssum species were used to detect ATP-PRT1 and ATP-PRT2 transcripts and heterologous A. thaliana cDNA probes to detect all other His biosynthesis genes. The specific radioactivity of the two ATP-PRT 3′-UTR probes was determined and for both genes was found to be slightly higher for the A. montanum probes (data not shown). Expression of β-tubulin transcript was used as a control for equal loading of RNA. The results shown are from one experiment representative of three for the ATP-PRT transcripts or representative of two for other transcripts.

Figure 5.

Elevated ATP-PRT Expression Is Correlated with Ni Tolerance in the Genus Alyssum. RNA gel blot analysis of ATP-PRT transcript abundance in root and shoot tissue of A. lesbiacum (Al), A. serpyllifolium (As), A. montanum (Am), and A. thaliana (At). Tissue was pooled from 10 6-week-old plants cultivated hydroponically without added Ni. Ten micrograms of total RNA was probed with an internal 415-bp fragment of the A. thaliana ATP-PRT2 cDNA. Equal loading of RNA was checked by ethidium bromide (EtBr) staining. The same expression pattern was observed when the corresponding A. thaliana ATP-PRT1 probe was used (data not shown).

Figure 6.

Ni-Induced Release of His from Roots into the Xylem of A. lesbiacum. Concentration of [³H]His in xylem exudates from 8-week-old hydroponically grown A. lesbiacum plants, cultivated in standard nutrient solution not containing Ni and then transferred to 300 μM NiSO₄ (Ni), 300 μM ZnSO₄ (Zn), or no addition (control) for 16 h. Xylem sap exudation rates were not significantly different under the three treatments (data not shown). Bars are means + se (n = 4 individual plants). The asterisk indicates significance at P < 0.05.

Figure 7.

DNA Gel Blot Analysis of ATP-PRT Copy Number in A. lesbiacum. Three micrograms of genomic DNA were digested with EcoRV (E; no site in either cDNA), XhoI (X; one site in ATP-PRT1 cDNA), or PstI (P; one site in both ATP-PRT1 and ATP-PRT2 cDNAs) and probed with a ³²P-labeled probe corresponding to the full-length ATP-PRT2 cDNA (minus predicted plastid transit peptide) under low-stringency conditions. The same banding pattern was observed when the corresponding ATP-PRT1 probe was used under the same conditions (data not shown).

Figure 8.

Maximum Likelihood Tree Depicting Phylogenetic Relationships between Different ATP-PRT cDNA Sequences in Plants. cDNA sequences for A. lesbiacum and A. montanum were obtained in this work. Sequences for A. thaliana (ATP-PRT1, AB025249; ATP-PRT2, AB025250), T. goesingense (AF003347), Oryza sativa (AC099399), and Zea mays (AY112299) were retrieved from GenBank. Full-length cDNA sequences for Medicago truncatula, Lycopersicon esculentum, Solanum tuberosum, Triticum aestivum, Hordeum vulgare, and Physcomitrella patens were assembled from ESTs as described in Methods. Sequences for the open reading frame of ATP-PRT were aligned using ClustalW and refined manually, but the sequence encoding the predicted N-terminal plastid transit peptide was excluded from the phylogenetic analysis (see Supplemental Figure 1 online). The angiosperm maximum likelihood tree was rooted on the bryophyte outgroup (P. patens). Bootstrap support values (as percentages of 1000 replicates) are given above nodes subtending the relevant branches. Inferred gene duplication events are indicated by vertical bars.

Figure 9.

A. lesbiacum ATP-PRT1 and ATP-PRT2 Encode Functional ATP-PRT Proteins. Functional complementation of the E. coli hisG⁻ mutant strain NK5526. Bacteria transformed with pUCmod:PRT1 or pUCmod:PRT2 were able to grow on minimal medium in the absence of supplemental 50 μM His, whereas those transformed with the empty vector (control) were not.

Figure 10.

Free His Concentrations in 35S:PRT2 Transgenic A. thaliana Are Correlated with Transgene Expression. (A) Free His concentration in rosette tissue of wild-type, empty-vector control (pBI121), and 35S:PRT2 transgenic lines was determined by HPLC analysis. Bars are means + se of three independent replicates per line, each consisting of tissue pooled from two 21-d-old plants. The 35S:PRT2 transgenic lines are ranked according to free His concentration. Eleven lines had significantly (P < 0.01) higher concentrations of free His than wild-type plants (line 2.1 onwards), as determined using the least significant difference test (Sokal and Rohlf, 1995). A. lesbiacum ATP-PRT2 transgene expression was quantified as phosphor imager counts. (B) RNA gel blot analysis of A. lesbiacum (Al) ATP-PRT2 expression in the 35S:PRT2 transgenic lines. Total RNA was isolated from rosette tissue pooled from six 21-d-old plants and probed with a 156-bp cDNA fragment encoding the predicted plastid transit peptide of the A. lesbiacum ATP-PRT2 protein. EtBr, ethidium bromide.

Figure 11.

35S:PRT2 His-Overproducing Lines Show Increased Tolerance to Ni. Dry biomass at harvest of shoots and roots of 5-week-old plants cultivated hydroponically for 3 weeks on 30 μM NiSO₄, expressed as a percentage of biomass of each line grown in the absence of added Ni. Bars are means + se (n = 12 plants, subdivided between four different culture vessels) for each line. Significant differences between wild-type and transgenic lines (i.e., with a probability below the threshold of 0.01 calculated using the Bonferroni method) are indicated with two asterisks for P < 0.01 and with three asterisks for P < 0.001. For the wild type, empty-vector control (pBI121), and lines 1.1, 22.1, 30.5, and 21.2, absolute shoot biomass in the absence of Ni was 26.7 ± 2.9 mg, 38.7 ± 3.4 mg, 28.8 ± 3.3 mg, 33.8 ± 3.9 mg, 22.6 ± 2.6 mg, and 32.1 ± 3.5 mg, respectively, and root biomass was 7.0 ± 0.7 mg, 8.8 ± 0.8 mg, 7.1 ± 1.0 mg, 7.5 ± 1.2 mg, 5.4 ± 0.6 mg, and 7.0 ± 0.8 mg, respectively. Lines 30.5 and 21.2 were shown to exhibit enhanced Ni tolerance relative to the controls in two additional independent experiments.

Figure 12.

35S:PRT2 His-Overproducing Lines Do Not Show Elevated Tissue Concentrations of Ni. Ni content of tissues was determined by atomic absorption spectrophotometry. Plants were exposed to 0, 10, 20, or 50 μM added Ni for 3 weeks before harvesting. Bars are means + se of four independent replicates per line, each replicate consisting of tissue pooled from four individual plants. (A) Shoot Ni concentration. (B) Root Ni concentration.

Figure 13.

Xylem Sap Concentration of His and Flux of Ni in 35S:PRT2 His-Overproducing Lines. Xylem sap for analysis was collected over a period of 12 h from detopped root systems. (A) His concentration in xylem sap of plants exposed to 0 or 10 μM Ni for 3 weeks before sampling. Bars are means + se (n = 3 plants) normalized to the total concentration of free amino acids measured in each sample. Significant differences between the transgenic lines and the wild type are indicated with one asterisk for P < 0.05 and with two asterisks for P < 0.01. (B) Ni flux in xylem sap of plants exposed to 1 or 10 μM Ni for 3 weeks before sampling. Values were calculated as the product of measured sap Ni concentration and sap flow rate and are expressed as means + se (n = 5 plants). Note the 10-fold scale expansion for 1 μM Ni treatment. Two-way analysis of variance yielded no evidence for a significant difference between lines or for a line–Ni treatment interaction (both P > 0.05).