Schwalm Katja, Aloni Roni, Langhans Markus, Heller Werner, Stich Susanne, Ullrich Cornelia I
Institut für Botanik, Technische Universität, Schnittspahnstrasse 3, 64287 Darmstadt, Germany.
Planta. 2003 Dec;218(2):163-78. doi: 10.1007/s00425-003-1104-6. Epub 2003 Oct 2.
Agrobacterium tumefaciens-induced plant tumors accumulate considerable concentrations of free auxin. To determine possible mechanisms by which high auxin concentrations are maintained, we examined the pattern of auxin and flavonoid distribution in plant tumors. Tumors were induced in transformants of Trifolium repens (L.), containing the beta-glucuronidase ( GUS)-fused auxin-responsive promoter ( GH3) or chalcone synthase ( CHS2) genes, and in transformants of Arabidopsis thaliana (L.) Heynh., containing the GUS-fused synthetic auxin response element DR5. Expression of GH3::GUS and DR5::GUS was strong in proliferating metabolically active tumors, thus suggesting high free-auxin concentrations. Immunolocalization of total auxin with indole-3-acetic acid antibodies was consistent with GH3::GUS expression indicating the highest auxin concentration in the tumor periphery. By in situ staining with diphenylboric acid 2-aminoethyl ester, by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and two-photon laser-scanning microscopy spectrometry, tumor-specific flavones, isoflavones and pterocarpans were detected, namely 7,4'-dihydroxyflavone (DHF), formononetin, and medicarpin. DHF was the dominant flavone in high free-auxin-accumulating stipules of Arabidopsis leaf primordia. Flavonoids were localized at the sites of strongest auxin-inducible CHS2::GUS expression in the tumor that was differentially modulated by auxin in the vascular tissue. CHS mRNA expression changes corresponded to the previously analyzed auxin concentration profile in tumors and roots of tumorized Ricinus plants. Application of DHF to stems, apically pretreated with alpha-naphthaleneacetic acid, inhibited GH3::GUS expression in a fashion similar to 1-N-naphthyl-phthalamic acid. Tumor, root and shoot growth was poor in inoculated tt4(85) flavonoid-deficient CHS mutants of Arabidopsis. It is concluded that CHS-dependent flavonoid aglycones are possibly endogenous regulators of the basipetal auxin flux, thereby leading to free-auxin accumulation in A. tumefaciens-induced tumors. This, in turn, triggers vigorous proliferation and vascularization of the tumor tissues and suppresses their further differentiation.
根癌农杆菌诱导的植物肿瘤积累了相当浓度的游离生长素。为了确定维持高生长素浓度的可能机制，我们研究了植物肿瘤中生长素和黄酮类化合物的分布模式。在含有β-葡萄糖醛酸酶（GUS）融合的生长素响应启动子（GH3）或查尔酮合酶（CHS2）基因的白三叶草（Trifolium repens，L.）转化体中，以及在含有GUS融合的合成生长素反应元件DR5的拟南芥（Arabidopsis thaliana，L.）Heynh.转化体中诱导肿瘤形成。GH3::GUS和DR5::GUS在增殖的代谢活跃肿瘤中表达强烈，因此表明游离生长素浓度较高。用吲哚-3-乙酸抗体对总生长素进行免疫定位与GH3::GUS表达一致，表明肿瘤周边生长素浓度最高。通过用2-氨基乙基二苯基硼酸原位染色、薄层色谱、反相高效液相色谱和双光子激光扫描显微镜光谱法，检测到肿瘤特异性黄酮、异黄酮和紫檀素，即7,4'-二羟基黄酮（DHF）、芒柄花素和苜蓿素。DHF是拟南芥叶原基高游离生长素积累托叶中的主要黄酮。黄酮类化合物定位于肿瘤中生长素诱导最强的CHS2::GUS表达位点，该位点在维管组织中受到生长素的差异调节。CHS mRNA表达变化与先前分析的肿瘤化蓖麻植物肿瘤和根中的生长素浓度分布一致。将DHF应用于用α-萘乙酸预处理过的茎尖，以类似于1-N-萘基邻苯二甲酸的方式抑制GH3::GUS表达。在接种了拟南芥tt4(85)黄酮类缺陷型CHS突变体的植株中，肿瘤、根和茎的生长较差。得出的结论是，CHS依赖性黄酮苷元可能是生长素向基运输通量的内源性调节因子，从而导致根癌农杆菌诱导的肿瘤中游离生长素积累。这反过来又触发肿瘤组织的旺盛增殖和血管化，并抑制其进一步分化。
