Invertases involved in the development of the parasitic plant Phelipanche ramosa: characterization of the dominant soluble acid isoform, PrSAI1

Abstract

Phelipanche ramosa L. parasitizes major crops, acting as a competitive sink for host photoassimilates, especially sucrose. An understanding of the mechanisms of sucrose utilization in parasites is an important step in the development of new control methods. Therefore, in this study, we characterized the invertase gene family in P. ramosa and analysed its involvement in plant development. Invertase-encoded cDNAs were isolated using degenerate primers corresponding to highly conserved regions of invertases. In addition to enzyme assays, gene expression was analysed using real-time quantitative reverse transcriptase-polymerase chain reaction during overall plant development. The dominant isoform was purified and sequenced using electrospray ionization-liquid chromatography-tandem mass spectrometry (ESI-LC-MS/MS). Five invertase-encoded cDNAs were thus characterized, including PrSai1 which encodes a soluble acid invertase (SAI). Of the five invertases, PrSai1 transcripts and SAI activity were dominant in growing organs. The most active invertase corresponded to the PrSai1 gene product. The purified PrSAI1 displayed low pI and optimal pH values, specificity for β-fructofuranosides and inhibition by metallic ions and competitive inhibition by fructose. PrSAI1 is a typical vacuolar SAI that is actively involved in growth following both germination and attachment to host roots. In addition, germinated seeds displayed enhanced cell wall invertase activity (PrCWI) in comparison with preconditioned seeds, suggesting the contribution of this activity in the sink strength of infected roots during the subsequent step of root penetration. Our results show that PrSAI1 and, possibly, PrCWI constitute good targets for the development of new transgenic resistance in host plants using proteinaceous inhibitors or silencing strategies.

Figure 1

Dendrogram of invertase amino acid sequences from various plants including sequences deduced from Phelipanche ramosa full‐length invertase genes. The neighbour‐joining consensus tree was inferred from 500 bootstrap replicates. Branches occurring in less than 50% bootstrap replicates were collapsed.

Figure 2

Development‐related changes in the levels of invertase‐encoded gene transcripts and invertase activities in Phelipanche ramosa: (a, b) independent development following seed germination; PS, preconditioned seeds; GS, germinated seeds; (c, d) parasitic stages following attachment to tomato roots. See Fig. 5 for abbreviations of the various plant organs. (a, c) Transcript accumulation expressed relative to elongation factor 1‐α gene (EF1α) transcript levels. Data are means ± standard error (n= 3). For each gene, values with the same letter are not significantly different (anova, SNK test, P < 0.05). (b, d) Invertase activities. Data are means ± standard error (n= 6). For each isoform, activities with the same letter (plain typeface) are not significantly different (anova, SNK test, P < 0.05). For each stage of development and organ, activities with the same letter (italic and bold typeface) are not significantly different (anova, SNK test, P < 0.05).

Figure 3

Tissue localization of soluble acid invertase (SAI) activity in seeds and flowering shoots of Phelipanche ramosa parasitizing tomato plants. (a1, b1) Control cross‐sections of basal (Fp.V) and apical (FS.V) shoots incubated after fixation in reaction medium without sucrose. (a2, b2) Assay sections incubated after fixation in a reaction mixture containing sucrose. SAI activity () is shown in blue in medullar and cortical parenchyma. No cell wall invertase (CWI) activity was detected on unfixed sections (data not shown). Staining on fixed sections was thus attributed to SAI. (a3, b3) Post‐activity staining of cellulosic (purple) and lignified (green) tissues using Mirande's reagent. (c) Germinated seeds incubated in the reaction mixture containing sucrose. (d1) NTB staining of seeds without sucrose in reaction medium. (d2) NTB staining of seeds with sucrose in reaction medium. (d3) Zoom of the radicle region stained with NTB: accumulation of reducing sugars produced from SAI‐mediated hydrolysis of sucrose is shown in black in the apex of the radicle. CP, cortical parenchyma; CT, conductive tissues; EH, epidermal hairs; MP, medullar parenchyma; NTB, nitrotetrazolium blue; Rd, radicle; SC, seed coat.

Figure 4

Amino acid sequence as deduced from the PrSai1 gene and amino acid sequences of the peptides generated by trypsin digestion of the soluble acid invertase (SAI) isoform purified from the subterranean shoots of Phelipanche ramosa parasitizing tomato plants. Peptides matching the putative P. ramosa acid invertase sequence are shaded, and tryptic cleavage sites are indicated by arrows. The β‐fructofuranosidase motif and a highly conserved peptide domain with a catalytic cysteine residue are underlined (Sturm, 1999).

Figure 5

Developmental stages in Phelipanche ramosa parasitizing tomato plants: (a) stage III, growing tubercle (Tub.III); (b) stage IV, tubercle (Tub.IV) bearing the growing subterranean shoot (S.IV); (c) stage V, tubercle (Tub.V) bearing the emerged flowering shoot. Apical part of the flowering shoot is growing (FS.V) and bears fruits containing developing seeds (F.V). Basal part (Bp.V) does not bear flowers and is larger and more fibrous than the flowering shoot. HR, host roots (tomato). Bar, 1 cm.