Molecular phenotyping of the pal1 and pal2 mutants of Arabidopsis thaliana reveals far-reaching consequences on phenylpropanoid, amino acid, and carbohydrate metabolism

Abstract

The first enzyme of the phenylpropanoid pathway, Phe ammonia-lyase (PAL), is encoded by four genes in Arabidopsis thaliana. Whereas PAL function is well established in various plants, an insight into the functional significance of individual gene family members is lacking. We show that in the absence of clear phenotypic alterations in the Arabidopsis pal1 and pal2 single mutants and with limited phenotypic alterations in the pal1 pal2 double mutant, significant modifications occur in the transcriptome and metabolome of the pal mutants. The disruption of PAL led to transcriptomic adaptation of components of the phenylpropanoid biosynthesis, carbohydrate metabolism, and amino acid metabolism, revealing complex interactions at the level of gene expression between these pathways. Corresponding biochemical changes included a decrease in the three major flavonol glycosides, glycosylated vanillic acid, scopolin, and two novel feruloyl malates coupled to coniferyl alcohol. Moreover, Phe overaccumulated in the double mutant, and the levels of many other amino acids were significantly imbalanced. The lignin content was significantly reduced, and the syringyl/guaiacyl ratio of lignin monomers had increased. Together, from the molecular phenotype, common and specific functions of PAL1 and PAL2 are delineated, and PAL1 is qualified as being more important for the generation of phenylpropanoids.

Figure 1.

Phenotypes of the Wild-Type C24 and pal1, pal2, and pal1 pal2 Mutants. Lignin staining in the basal part of 3-month-old inflorescence stems ([A] to [L]) and silique phenotype (M). (A) The wild type stained with aniline sulfate. (B) pal1 mutant stained with aniline sulfate. (C) pal2 mutant stained with aniline sulfate. (D) pal1 pal2 mutant stained with aniline sulfate. (E) The wild type stained with KMnO₄:HCl:NH₃. (F) pal1 mutant stained with KMnO₄:HCl:NH₃. (G) pal2 mutant stained with KMnO₄:HCl:NH₃. (H) pal1 pal2 mutant stained with KMnO₄:HCl:NH₃. (I) The wild type stained with phloroglucinol:HCl. (J) pal1 mutant stained with phloroglucinol:HCl. (K) pal2 mutant stained with phloroglucinol:HCl. (L) pal1 pal2 mutant stained with phloroglucinol:HCl. (M) Siliques of the wild type (bottom) and pal1 pal2 mutant (top) at seed maturity of the wild type.

Figure 2.

Cell Wall Ultrastructure in the Basal Part of 3-Month-Old Inflorescence Stems of the Wild Type and pal1 pal2 Mutants. (A) Xylem region of the wild type. (B) Xylem region of pal1 pal2 mutants. Arrows point to aberrantly shaped walls and to regions punctured with little holes. (C) and (D) Cell walls in the xylem region of pal1 pal2 mutants. In (D), loosened cellulose fibrils become apparent. (E) Interfascicular fiber region of the wild type. (F) Interfascicular fiber region of pal1 pal2 mutants. Arrow points to whiter cell corners. (G) and (H) Cell walls in the interfascicular fiber region of pal1 pal2 mutants. Bar in (A), (B), (E), and (F) = 2.5 μm; bar in (C), (D), (G), and (H) = 1 μm.

Figure 3.

Expression of PAL Genes in 10-Week-Old Inflorescence Stems of the Wild Type and pal1, pal2, and pal1 pal2 Mutants. Semiquantitative RT-PCRs were performed on a pool of 10 individuals of each respective genotype (see Methods). The experiment was repeated with similar results.

Figure 4.

PAL Activity in 10-Week-Old Inflorescence Stems of the Wild Type and pal1, pal2, and pal1 pal2 Mutants. PAL activity of crude extracts was determined in inflorescence stems (n = 10) and is expressed as average PAL activity in pmol cinnamic acid (CA) s⁻¹ μg⁻¹ protein with standard deviation.

Figure 5.

Expression of the Monolignol Biosynthesis Genes in pal Mutants. Genes (34) were annotated with sensitive annotation methods for the 10 enzymes currently known to participate in the generation of monolignols (Raes et al., 2003). Semiquantitative RT-PCRs were performed on two pools of 10 inflorescence stems of each respective genotype (see Methods). Expression levels were normalized to ACT2 gene expression before calculation of fold changes in expression levels taking the wild-type level as reference. Bars represent the average expression level of two biological repeats. Lines mark the level of fourfold upregulation or downregulation. Note that the following genes were not found to be expressed at this stage of inflorescence stem development (10-week-old plants): C4H, C3H2, C3H3, F5H2, CAD1, CAD4, and CAD5. For primers and gene nomenclature, see Raes et al. (2003).

Figure 6.

Number and Functional Categories of Genes with Altered Expression in pal1, pal2, and pal1 pal2 Mutants as Compared with the Wild Type. (A) Summary from both cDNA-AFLP and microarray analyses (Tables 1 and 2) corrected for the genes that were revealed by both methods. (B) Functional categories of the groups of genes controlled either by pal1, pal2, or both. The group pal1 comprises genes differentially expressed in only pal1 (n = 20) or pal1 and the double mutant (n = 5). The group pal2 comprises genes differentially expressed in only pal2 (n = 29) or pal2 and the double mutant (n = 10). The group pal1 and pal2 comprises genes differentially expressed in both single mutants but not in the double mutant (n = 5), genes expressed in pal1, pal2, and the double mutant (n = 27), and genes expressed exclusively in the double mutant (n = 1). AA, amino acid metabolism; CH, carbohydrate metabolism; LI, light related; M, miscellaneous; PP, phenylpropanoid related; ST, signal transduction; STR, stress response; TRP, transport; UN, unknown.

Figure 7.

Integration of the Metabolic Pathways Affected by a Decreased Carbon Flux into the Phenylpropanoid Pathway, as Evidenced by Molecular Alterations in pal Mutants. Results of the transcriptome analyses (RT-PCR, cDNA-AFLP, and microarray) and the biochemical analyses (extractable phenolics, amino acids, and lignin) are incorporated. A dashed line indicates transport into another compartment; right and left harpoons mark a reversible enzyme reaction and arrows an irreversible enzyme reaction, respectively; question mark denotes a not fully established path. Arrows pointed up or down indicate the upregulation or downregulation of the gene or metabolite, respectively. The following color code is used: red, transcripts and metabolites linked with the pal1 but not the pal2 mutation; green, transcripts and metabolites linked with the pal2 but not the pal1 mutation; orange, transcripts and metabolites linked with the pal1 and pal2 mutations; orange with asterisk, transcripts and metabolites occurring only in the pal1 pal2 double mutant. Abbreviations are listed per pathway. Carbohydrate metabolism: 1,3-BisPGA, 1,3-bisphosphoglycerate; BXL, β-xylosidase; CESA, cellulose synthase; DHAP, dihydroxyacetone phosphate; Ery4P, erythrose 4-phosphate; F1,6-BisP, fructose 1,6-bisphosphate; F6P, fructose 6-phosphate; G1P, glucose 1-phosphate; G6P, glucose 6-phosphate; GAP, glyceraldehyde 3-phosphate; INV, invertase; PEP, phospho-enol-pyruvate; 2-PGA, 2-phosphoglycerate; 3-PGA, 3-phosphoglycerate; 3-PGAkin, 3-phosphoglycerate kinase; R1,5-BisP, ribulose 1,5-bisphosphate; RubAct, Rubisco activase; StP, starch phosphorylase; StS, starch synthase; TK, transketolase; TPI, triose-phosphate isomerase; TPS, trehalose 6-phosphate synthase. Jasmonate biosynthesis: AOS, allene oxide synthase; 3FAD, ω-3 fatty acid desaturase; JA, jasmonate. Methionine metabolism: CysS, cystathionine β-lyase (Cys synthase); MetS, Met synthase. Shikimate pathway: DAHP, 3-deoxy-d-arabino-heptulosonate 7-phosphate; Ery4P, erythrose 4-phosphate; 4-HBA, para-hydroxybenzoate; 4-HBald, para-hydroxybenzaldehyde; PEP, phospho-enol-pyruvate; UbiRed, NADH-ubiquinone oxidoreductase. Recycling of NH₄⁺¹: ASN3, Asn synthase 3. Phenylpropanoid pathway: ATR3, NADPH-ferrihemoprotein reductase 3; CCoAOMT, caffeoyl-CoA 3-O-methyltransferase; 4-HBA, para-hydroxybenzoate. Monolignol biosynthesis: G lignin, guaiacyl lignin monomers; S lignin, syringyl lignin monomers. Flavonoid biosynthesis: CHS, chalcone synthase.

Figure 8.

Profiles of Soluble Phenolic Metabolites in 3-Month-Old Inflorescence Stems of the Wild-Type C24, pal1, and pal2, pal1 pal2 Mutants. Soluble phenolics are separated by reverse-phase HPLC. The maxplot chromatograms are given as absorbance units at 200 to 450 nm and are scaled down to show the differences in the most abundant peaks. A, B, and C denote the three major kaempferol glycosides decreased in the double mutant.

Figure 9.

Identification by Mass Spectrometry of Five Soluble Phenolics Significantly Altered in pal Mutants. All MS spectra are given as relative abundance of molecules with a scale to 100%. (A) Full MS spectra obtained in the positive ionization mode for the three major peaks found to be altered significantly in pal mutants (indicated with A, B, and C in Figure 8). The mass-to-charge (m/z) values in the MS spectra for A, B, and C confirm that these compounds are hexose-conjugated kaempferols. The A in Figure 8 corresponds to kaempferol 3-O-β-[β-d-glucopyranosyl (1-6)-d-glucopyranoside]-7-O-α-l-rhamnopyranoside, B to kaempferol 3-O-β-d-glucopyranoside-7-O-α-l-rhamnopyranoside, and C to kaempferol 3-O-α-l-rhamnopyranoside-7-O-α-l-rhamnopyranoside. (B) Identification of the HPLC peak at RT 7.331 min as scopolin by full MS in the negative ionization mode, MS² of m/z 413, and MS³ of m/z 191. (C) Identification of the HPLC peak at RT 11.178 min as feruloyl malate coupled 4–O–8 to coniferyl alcohol by full MS, MS² of m/z 505, and MS³ of m/z 389. The MS⁴ of m/z 193, with the characteristic fragmentation pattern of ferulic acid, is not shown. (D) Identification of the HPLC peak at RT 15.119 min as feruloyl malate coupled 5–8 to coniferyl alcohol by full MS, MS² of m/z 487, and MS³ of m/z 371. Fragmentation of m/z 371 yielded peaks at m/z 353, 341, and 338, in abundances reminiscent of a phenylcoumaran (K. Morreel and W. Boerjan, unpublished data).