Rea Giuseppina, de Pinto Maria Concetta, Tavazza Raffaela, Biondi Stefania, Gobbi Valentina, Ferrante Paola, De Gara Laura, Federico Rodolfo, Angelini Riccardo, Tavladoraki Paraskevi
Department of Biology, Università degli Studi Roma Tre, Rome 00146, Italy.
Plant Physiol. 2004 Apr;134(4):1414-26. doi: 10.1104/pp.103.036764. Epub 2004 Apr 2.
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.
为了测试通过影响多胺分解代谢途径来改变多胺水平的可行性，我们获得了组成型表达玉米（Zea mays）多胺氧化酶（MPAO）或豌豆（Pisum sativum）铜胺氧化酶（PCuAO）的转基因烟草（Nicotiana tabacum）植株，这两种酶都是细胞外且能产生H₂O₂的酶。尽管转基因在细胞外空间有高表达水平，但纯合转基因植株中游离多胺的量与野生型植株相似，这表明要么多胺水平受到严格调控，要么两种重组蛋白与大量内源性多胺存在不同的区室化分布。此外，与未转化植株相比，转基因植株在木质化水平和植物形态上未观察到变化，而活性氧清除能力有微小但显著的变化得到了验证。MPAO和PCuAO烟草转基因植株仅在添加外源酶底物时才产生大量H₂O₂。这些观察结果证明了生理条件下烟草植株细胞外空间中可自由利用的多胺量有限，未转化的玉米和豌豆植株也进一步证实了这一点。MPAO转基因植株细胞悬液中外源添加多胺产生的H₂O₂量足以诱导程序性细胞死亡，这种死亡对过氧化氢酶处理敏感，且需要基因表达和类半胱天冬酶活性。MPAO和PCuAO转基因植株是研究细胞外空间中多胺分泌和缀合，以及确定多胺分解代谢在细胞壁发育和应激反应中实际何时以及如何发挥作用的优秀工具。
