Ectopic expression of maize polyamine oxidase and pea copper amine oxidase in the cell wall of tobacco plants

Abstract

To test the feasibility of altering polyamine levels by influencing their catabolic pathway, we obtained transgenic tobacco (Nicotiana tabacum) plants constitutively expressing either maize (Zea mays) polyamine oxidase (MPAO) or pea (Pisum sativum) copper amine oxidase (PCuAO), two extracellular and H(2)O(2)-producing enzymes. Despite the high expression levels of the transgenes in the extracellular space, the amount of free polyamines in the homozygous transgenic plants was similar to that in the wild-type ones, suggesting either a tight regulation of polyamine levels or a different compartmentalization of the two recombinant proteins and the bulk amount of endogenous polyamines. Furthermore, no change in lignification levels and plant morphology was observed in the transgenic plants compared to untransformed plants, while a small but significant change in reactive oxygen species-scavenging capacity was verified. Both the MPAO and the PCuAO tobacco transgenic plants produced high amounts of H(2)O(2) only in the presence of exogenously added enzyme substrates. These observations provided evidence for the limiting amount of freely available polyamines in the extracellular space in tobacco plants under physiological conditions, which was further confirmed for untransformed maize and pea plants. The amount of H(2)O(2) produced by exogenously added polyamines in cell suspensions from the MPAO transgenic plants was sufficient to induce programmed cell death, which was sensitive to catalase treatment and required gene expression and caspase-like activity. The MPAO and PCuAO transgenic plants represent excellent tools to study polyamine secretion and conjugation in the extracellular space, as well as to determine when and how polyamine catabolism actually intervenes both in cell wall development and in response to stress.

Figure 1.

Generation and molecular characterization of transgenic tobacco plants expressing MPAO or PCuAO. A, Map of the MPAO-Ω-pBI and PCuAO-pBI constructs used for Agrobacterium-mediated transformation of tobacco plants. Primers (Pr/Pf and Dr/Df) used for PCR analysis and lengths of amplified fragments are indicated. B, PCR analysis of alkali-treated leaf pieces from tobacco plants transformed with the MPAO-Ω-pBI and PCuAO-pBI constructs. Numbers represent independent MPAO and PCuAO transgenic lines. C+, positive control using purified MPAO or PCuAO cDNA; C1 and C2, negative controls using leaf pieces from untransformed plants and MPAO- or PCuAO-specific primers, respectively; M, M_r marker (1-kb DNA ladder; Life Technologies/Gibco-BRL, Cleveland).

Figure 2.

Expression levels of MPAO and PCuAO in different transgenic tobacco lines. Expression levels were determined by enzyme activity assays (top) and western-blot analysis (bottom) in crude leaf extracts. Values are mean ± se from three replicates. Numbers represent independent MPAO and PCuAO transgenic lines. WT, untransformed tobacco plants; C⁺, purified MPAO or PCuAO protein. For western-blot analysis, extracts were normalized for the amount of total soluble proteins.

Figure 3.

Detection of H₂O₂ production in plant tissues of tobacco, maize, and pea plants on KI- and starch-containing medium. A, Leaf discs, stem sections, and roots of untransformed plants (WT) and MPAO- or PCuAO-expressing tobacco transgenic plants were placed in the medium in the absence (−Spd/Put) or in the presence of 1 mm Spd (+Spd) or Put (+Put). B, Leaf discs from maize and pea plants were placed in the medium in the absence (−Spd; −Put) and in the presence of Spd (+Spd) or Put (+Put) at a final concentration of 1 mm. Color development was allowed for 24 h.

Figure 4.

In situ detection of H₂O₂ production by the DAB-uptake method in the MPAO and PCuAO transgenic tobacco plants. Leaves from wild-type (B, D, and G) and MPAO (A and E) or PCuAO (C and F) transgenic plants were supplied with DAB for 18 h in the absence (E–G) and in the presence of 1 mm Spd (A and B) or 1 mm Put (C and D).

Figure 5.

Steady-state accumulation of H₂O₂ in cell suspensions from MPAO transgenic plants. Kinetics of H₂O₂ accumulation in the culture medium of cell suspensions obtained from wild-type (C) or MPAO transgenic (T) plants was determined spectrophotometrically after addition or not of 2 mm or 6 mm Spd. Data are from a single representative experiment, which was repeated in triplicate. The inset shows the remaining amount of Spd in the medium of MPAO cell suspensions at various time intervals after addition of 6 mm Spd.

Figure 6.

H₂O₂ scavenging in tobacco cell suspensions. Degradation of exogenously added H₂O₂ (0.35 mm) was determined in wild-type (W) and MPAO-expressing (T) cell suspensions in the absence (W; T) and in the presence (W + Spd) of 6 mm Spd. Data are from a single representative experiment, which was repeated in triplicate.

Figure 7.

Effect of H₂O₂ on cell viability of tobacco cell suspensions. Cell death was measured by Evans blue staining 24 h after addition of various amounts of Spd (0, 2 mm, and 6 mm) in wild-type (W) and MPAO-expressing (T) cell suspensions. Values are mean ± se (n = 5). Asterisks indicate values significantly different from those of cells mock treated with H₂O by one-way ANOVA test (P < 0.05).

Figure 8.

Induction of cell death by oxidative stress in tobacco cell suspensions. A, Kinetics of H₂O₂ accumulation induced by 6 mm Spd in MPAO-expressing cell suspensions. Cells were pretreated (Spd + Cat) or not (Spd) with catalase for 30 min before addition of Spd. Data are from a single representative experiment, which was repeated in triplicate. B, Cell death was determined by Evans blue staining in wild-type (W) and MPAO-expressing (T) cell suspensions 24 h after addition (+Spd) or not (−Spd) of 6 mm Spd. Cell death was also determined in cell suspensions pretreated with catalase for 30 min before addition of 6 mm Spd (+Spd+Cat). Each point represents mean value of three independent experiments.

Figure 9.

Induction of PCD by Spd-induced production of H₂O₂ in MPAO-expressing cell suspensions. Cell death was estimated by Evans blue staining in MPAO-expressing cell suspensions (T) 15 h after addition of 0, 2 mm, or 6 mm Spd. Where indicated, 40 μm cycloheximide (A) or 40 μm caspase inhibitor (B) was added to the culture medium 30 min prior to Spd administration. Values are mean ± se (n = 3). Asterisks indicate values significantly different from those of cells treated with the same amount of Spd and not with cycloheximide or caspase inhibitor P < 0.05 (by one-way ANOVA test).