A tomato peroxidase involved in the synthesis of lignin and suberin

Abstract

The last step in the synthesis of lignin and suberin has been proposed to be catalyzed by peroxidases, although other proteins may also be involved. To determine which peroxidases are involved in the synthesis of lignin and suberin, five peroxidases from tomato (Lycopersicon esculentum) roots, representing the majority of the peroxidase activity in this organ, have been partially purified and characterized kinetically. The purified peroxidases with isoelectric point (pI) values of 3.6 and 9.6 showed the highest catalytic efficiency when the substrate used was syringaldazine, an analog of lignin monomer. Using a combination of transgenic expression and antibody recognition, we now show that the peroxidase pI 9.6 is probably encoded by TPX1, a tomato peroxidase gene we have previously isolated. In situ RNA hybridization revealed that TPX1 expression is restricted to cells undergoing synthesis of lignin and suberin. Salt stress has been reported to induce the synthesis of lignin and/or suberin. This stress applied to tomato caused changes in the expression pattern of TPX1 and induced the TPX1 protein. We propose that the TPX1 product is involved in the synthesis of lignin and suberin.

Figure 1

IEF in the pH 8.0 to 10 range of extracts prepared from the aerial part of tobacco seedlings developed for peroxidase activity and cross-reacted with the antibodies against the peroxidase of pI 9.6. A, Peroxidase activity of the soluble and ionic extracts from transgenic (TPX1) and control tobacco lines. B, Western blot of the IEF gel of the ionic extract. One unit of enzyme is defined as the amount of enzyme forming 1 μmol product min⁻¹. The same activity (3 units) was loaded in each lane.

Figure 2

TPX1 expression in tomato roots by in situ hybridization. Transversal sections of hydroponically grown tomato roots were probed with digoxigenin-labeled antisense TPX1 RNA. A, Expression of TPX1 (RNA signal was detected as a dark brown or purple color) in the apical zone of the root (last 0.5 cm from the root tip). B, Detail of A showing that the expression is restricted to the endodermis (En) and protoxylem (P). C, Expression of TPX1 (purple) in the medium zone of the root (8–10 cm from the root tip). D, Detail of C showing the expression in the endodermis (En). E, Expression of TPX1 in the basal zone of the root (13–15 cm from the root tip). F, Detail of E revealing expression in the endodermis (En) and the exodermis (Ex).

Figure 3

Tissue printing of hand-sectioned tomato roots made to react with the antibodies raised against the pI 9.6 isoenzyme. A, Control. B, Salinized. En, Endodermis; Ep, epidermis; VC, vascular cylinder.

Figure 4

Analysis of TPX1 expression in root sections of tomato plants treated with 100 mm NaCl for 24 h. A, Expression of TPX1 (dark brown) in the apical zone of the root (last 0.5 cm from the root tip). B, Detail of A showing that the expression is completely abolished in the endodermis and protoxylem after NaCl treatment. C, Expression of TPX1 (purple) in the medium zone of the root (8–10 cm from the root tip). D, Detail of C showing TPX1 expression in the endodermis (En). E, Expression of TPX1 in the basal zone of the root (13–15 cm from the root tip). F, Detail of E revealing that TPX1 expression is limited to the endodermis (En).

Figure 5

Western blot of cationic PAGE of tomato root extracts at different days after NaCl treatment. C, Control; S, salinized. Peroxidases of R_F values of 0.26 and 0.32 correspond to the 8.2 and 9.6 pI isoforms, respectively.