State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin, 150040, China.
College of Life Science, Northeast Forestry University, Harbin, 150040, China.
Tree Physiol. 2020 Apr 8;40(4):520-537. doi: 10.1093/treephys/tpaa008.
The copper chaperone ATX1 has been investigated previously in the herbaceous plants Arabidopsis and rice. However, the molecular mechanisms of ATX1 underlying copper transport and functional characteristics in the woody plant Populus are poorly understood. In this study, PnATX1 and PnATX2 of Populus simonii × P. nigra were identified and characterized. Sequence analysis showed that PnATXs contained the metal-binding motif MXCXXC in the N-terminus and a lysine-rich region. Phylogenetic analysis of ATX protein sequences revealed that PnATXs were clustered in the same group as AtATX1. PnATX proteins were localized in the cytoplasm and nucleus. Tissue-specific expression analysis showed that PnATX1 and PnATX2 were expressed in all analyzed tissues and, in particular, expressed to a higher relative expression level in young leaves. Quantitative real-time PCR analysis indicated that each PnATX gene was differentially expressed in different tissues under treatments with copper, zinc, iron, jasmonate and salicylic acid (SA). The copper-response element GTAC, methyl jasmonate and salicylic acid responsiveness elements and other cis-acting elements were identified in the PnATX1 and PnATX2 promoters. Expression of β-glucuronidase driven by the PnATX1 promoter was observed in the apical meristem of 7-day-old Arabidopsis transgenic seedlings, and the signal strength was not influenced by deficient or excessive copper conditions. Both PnATX1 and PnATX2 functionally rescued the defective phenotypes of yeast atx1Δ and sod1Δ strains. Under copper excess and deficiency conditions, transgenic Arabidopsis atx1 mutants harboring 35S::PnATX constructs exhibited root length and fresh weight similar to those of the wild type and higher than those of Arabidopsis atx1 mutants. Superoxide dismutase activity decreased in transgenic lines compared with that of atx1 mutants, whereas peroxidase and catalase activities increased significantly under excess copper. The results provide a basis for elucidating the role of Populus PnATX genes in copper homeostasis.
铜伴侣蛋白 ATX1 先前已在草本植物拟南芥和水稻中进行了研究。然而，在木本植物杨树中，ATX1 对铜运输的分子机制及其功能特性仍知之甚少。本研究鉴定并表征了黑杨×青杨的 PnATX1 和 PnATX2。序列分析表明，PnATXs 在 N 端含有金属结合基序 MXCXXC 和富含赖氨酸的区域。ATX 蛋白序列的系统发育分析表明，PnATXs 与 AtATX1 聚类在同一组。PnATX 蛋白定位于细胞质和细胞核。组织特异性表达分析表明，PnATX1 和 PnATX2 在所有分析的组织中均有表达，特别是在幼叶中表达相对较高。定量实时 PCR 分析表明，在铜、锌、铁、茉莉酸和水杨酸（SA）处理下，每个 PnATX 基因在不同组织中的表达均不同。在 PnATX1 和 PnATX2 启动子中鉴定到 GTAC 铜反应元件、茉莉酸甲酯和水杨酸响应元件以及其他顺式作用元件。在 7 天大的拟南芥转基因幼苗的顶端分生组织中观察到由 PnATX1 启动子驱动的β-葡萄糖醛酸酶的表达，信号强度不受铜缺乏或过量条件的影响。PnATX1 和 PnATX2 均可以挽救酵母 atx1Δ 和 sod1Δ 菌株的缺陷表型。在铜过量和缺乏条件下，携带 35S::PnATX 构建体的 atx1 突变体拟南芥的根长和鲜重与野生型相似，高于 atx1 突变体拟南芥。与 atx1 突变体相比，转基因拟南芥中超氧化物歧化酶活性降低，而过氧化物酶和过氧化氢酶活性在铜过量时显著增加。这些结果为阐明杨树 PnATX 基因在铜稳态中的作用提供了依据。
