Deciphering the role of aspartate and prephenate aminotransferase activities in plastid nitrogen metabolism

Abstract

Chloroplasts and plastids of nonphotosynthetic plant cells contain two aspartate (Asp) aminotransferases: a eukaryotic type (Asp5) and a prokaryotic-type bifunctional enzyme displaying Asp and prephenate aminotransferase activities (PAT). We have identified the entire Asp aminotransferase gene family in Nicotiana benthamiana and isolated and cloned the genes encoding the isoenzymes with plastidic localization: NbAsp5 and NbPAT. Using a virus-induced gene silencing approach, we obtained N. benthamiana plants silenced for NbAsp5 and/or NbPAT. Phenotypic and metabolic analyses were conducted in silenced plants to investigate the specific roles of these enzymes in the biosynthesis of essential amino acids within the plastid. The NbAsp5 silenced plants had no changes in phenotype, exhibiting similar levels of free Asp and glutamate as control plants, but contained diminished levels of asparagine and much higher levels of lysine. In contrast, the suppression of NbPAT led to a severe reduction in growth and strong chlorosis symptoms. NbPAT silenced plants exhibited extremely reduced levels of asparagine and were greatly affected in their phenylalanine metabolism and lignin deposition. Furthermore, NbPAT suppression triggered a transcriptional reprogramming in plastid nitrogen metabolism. Taken together, our results indicate that NbPAT has an overlapping role with NbAsp5 in the biosynthesis of Asp and a key role in the production of phenylalanine for the biosynthesis of phenylpropanoids. The analysis of NbAsp5/NbPAT cosilenced plants highlights the central role of both plastidic aminotransferases in nitrogen metabolism; however, only NbPAT is essential for plant growth and development.

Figure 1.

Asp and aromatic amino acid biosynthetic pathways in plants. A, Postchorismate pathway leading to the biosynthesis of aromatic amino acids. CM, Chorismate mutase; E4P, d-erythrose 4-phosphate; 4-HPP, 4-hydroxyphenylpyruvate; PEP, phosphoenolpyruvate; PPY, phenylpyruvate. B, Asp-derived amino acid biosynthesis. ALS, Acetolactate synthase; AS, Asn synthetase; ASN, asparaginase; BCAT, branched-chain aminotransferase; CBL, cystathionine β-lyase; CGS, cystathionine γ-synthase; DAP-D, diaminopimelate decarboxylase; DAP-F, diaminopimelate epimerase; DHAD, dihydroxy acid dehydratase; DHDPR, dihydrodipicolinate reductase; DHDPS, dihydrodipicolinate synthase; HSDH, homoserine dehydrogenase; HSK, homoserine kinase; KARI, ketol-acid reductoisomerase; MS, Met synthase; TD, Thr deaminase; TS, Thr synthase.

Figure 2.

Phenotypes of N. benthamiana plants subjected to VIGS of NbAsp5 and/or NbPAT genes. Photographs of wild-type (WT) and control (pTRVGW-EV) plants are shown in the top row. Phenotypes of 6-week-old plants silenced for NbAsp5, NbPAT, or NbAsp-5/NbPAT are shown in the bottom row. Similar phenotypes were observed in six different experiments, each with five biological replicates. All photographs were taken under the same magnification.

Figure 3.

Molecular characterization of VIGS of NbPAT and/or NbAsp5 genes in N. benthamiana leaves. A, Relative NbAsp5 gene expression in silenced plants compared with controls. At bottom, AAT activity is shown on native gels with protein extracts prepared from control and silenced plants. B, Relative NbPAT gene expression in control and silenced plants. At bottom, AAT activity and PAT activity are shown in protein extracts prepared from control and silenced plants resolved by native gel electrophoresis; PAT protein levels were analyzed by western blotting using anti-PAT specific antibodies (de la Torre et al., 2006). The expression levels for all genes were normalized to that of NbActin2. Error bars represent se. Asterisks indicate significant differences compared with control plants. AAT activity corresponding to NbASP5 and NbPAT on the gels was determined using extracts from N. benthamiana leaves (Fig. 2) according to a previously described protocol (de la Torre et al., 2007).

Figure 4.

Lignin content and histological lignin staining in N. benthamiana plants. A, Quantification of lignin in leaves (left) and stems (right) from N. benthamiana plants. WT, Wild type; pTRVGW-EV, control vector; pTRV-Asp5, silenced NbAsp5; pTRV-PAT, silenced NbPAT; pTRV-Asp5/PAT, cosilenced NbAsp5/NbPAT. Six independent biological replicates were measured. Average values and sd were calculated. Error bars represent se, and asterisks indicate significant differences compared with control plants. B, Histological detection of lignin in fresh-cut stem sections of N. benthamiana corresponding to control and silenced plants. Red-violet color shows the reaction of phloroglucinol-HCl with cinnamaldehyde end groups of lignin. [See online article for color version of this figure.]

Figure 5.

Chlorophyll content in leaves of N. benthamiana plants. Chlorophyll a and b contents in N. benthamiana leaves were determined as described in “Materials and Methods.” For each measurement, the average value and sd were calculated using five independent biological replicates. Error bars represent se. Asterisks indicate significant differences compared with control plants. FW, Fresh weight; WT, wild type.

Figure 6.

Amino acid content in plants silenced for NbPAT and/or NbAsp5. Amino acid profiles are shown in leaves from N. benthamiana silenced for NbPAT, NbAsp5, or both NbPAT and NbAsp5 compared with empty-vector controls (EV). Amino acid content was determined using an HPLC method with a UV detector developed for the determination of individual amino acids. A, Asp, Glu, Asn, and Gln. B, Lys, Thr, and Ile. C, Phe and Tyr. Data represent means of five plants. Significant differences between the control and silenced plants based on Student’s t test (P ≤ 0.05) are indicated with asterisks. FW, Fresh weight.

Figure 7.

qPCR expression analysis in plants silenced for NbPAT and/or NbAsp5. Total RNA was extracted from N. benthamiana leaves as described. Transcript levels were determined by qPCR. A, NbAK1, NbASDH1, and NbLL-DAP. B, NbPDH1 and NbADH1. C, NbPDT1, NbPDT2, and NbAAAT. D, NbPAL1, NbC4H, and NbANTS1. The expression level for all genes was normalized to that of NbActin2. Values represent means of two assays of real-time qPCR analysis, with four biological replicates in each. Error bars represent se. Asterisks indicate significant differences compared with control plants.

Figure 8.

Schematic representation of major metabolic effects of plastidic AAT and PAT silencing. VIGS resulted in altered amino acid profiles and altered expression of genes encoding enzymes involved in amino acid biosynthesis. Arrows indicate transcriptional up- or down-regulation of the corresponding genes under NbPAT silencing in N. benthamiana leaves. The alternate pathway leading to the biosynthesis of Phe is shadowed in gray. AAAT, Aromatic amino acid aminotransferase; Aro, arogenate; AS, Asn synthetase; ASDH, Asp semialdehyde dehydrogenase; Asp-4S, l-Asp 4-semialdehyde; Cho, chorismate; Cinn, trans-cinnamate; CM, chorismate mutase; Cou, p-coumarate; E4P, d-erythrose 4-phosphate; 4-HPP, 4-hydroxyphenylpyruvate; OAA, oxaloacetate; 2-OG, 2-oxoglutarate; OpLH, o-phospho-l-homoserine; PEP, phosphoenolpyruvate; PPY, phenylpyruvate; Pre, prephenate; THDPA, l-2,3,4,5-tetrahydrodipicolinate. [See online article for color version of this figure.].