Nieuwenhuizen Niels J, Chen Xiuyin, Wang Mindy Y, Matich Adam J, Perez Ramon Lopez, Allan Andrew C, Green Sol A, Atkinson Ross G
The New Zealand Institute for Plant and Food Research Limited, Auckland 1025, New Zealand (N.J.N., X.C., M.Y.W., R.L.P., A.C.A., S.A.G., R.G.A.);School of Biological Sciences, University of Auckland, Auckland 1142, New Zealand (N.J.N., A.C.A.); andThe New Zealand Institute for Plant and Food Research Limited, Palmerston North 4442, New Zealand (A.J.M.)
The New Zealand Institute for Plant and Food Research Limited, Auckland 1025, New Zealand (N.J.N., X.C., M.Y.W., R.L.P., A.C.A., S.A.G., R.G.A.);School of Biological Sciences, University of Auckland, Auckland 1142, New Zealand (N.J.N., A.C.A.); andThe New Zealand Institute for Plant and Food Research Limited, Palmerston North 4442, New Zealand (A.J.M.).
Plant Physiol. 2015 Apr;167(4):1243-58. doi: 10.1104/pp.114.254367. Epub 2015 Feb 3.
Two kiwifruit (Actinidia) species with contrasting terpene profiles were compared to understand the regulation of fruit monoterpene production. High rates of terpinolene production in ripe Actinidia arguta fruit were correlated with increasing gene and protein expression of A. arguta terpene synthase1 (AaTPS1) and correlated with an increase in transcript levels of the 2-C-methyl-D-erythritol 4-phosphate pathway enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS). Actinidia chinensis terpene synthase1 (AcTPS1) was identified as part of an array of eight tandemly duplicated genes, and AcTPS1 expression and terpene production were observed only at low levels in developing fruit. Transient overexpression of DXS in Nicotiana benthamiana leaves elevated monoterpene synthesis by AaTPS1 more than 100-fold, indicating that DXS is likely to be the key step in regulating 2-C-methyl-D-erythritol 4-phosphate substrate flux in kiwifruit. Comparative promoter analysis identified potential NAC (for no apical meristem [NAM], Arabidopsis transcription activation factor [ATAF], and cup-shaped cotyledon [CUC])-domain transcription factor) and ETHYLENE-INSENSITIVE3-like transcription factor (TF) binding sites in the AaTPS1 promoter, and cloned members of both TF classes were able to activate the AaTPS1 promoter in transient assays. Electrophoretic mobility shift assays showed that AaNAC2, AaNAC3, and AaNAC4 bind a 28-bp fragment of the proximal NAC binding site in the AaTPS1 promoter but not the A. chinensis AcTPS1 promoter, where the NAC binding site was mutated. Activation could be restored by reintroducing multiple repeats of the 12-bp NAC core-binding motif. The absence of NAC transcriptional activation in ripe A. chinensis fruit can account for the low accumulation of AcTPS1 transcript, protein, and monoterpene volatiles in this species. These results indicate the importance of NAC TFs in controlling monoterpene production and other traits in ripening fruits.
为了解果实单萜生成的调控机制，对两种萜烯谱不同的猕猴桃（中华猕猴桃属）进行了比较。成熟的软枣猕猴桃果实中萜品油烯的高生成率与软枣猕猴桃萜烯合酶1（AaTPS1）基因和蛋白表达的增加相关，并且与2-C-甲基-D-赤藓糖醇-4-磷酸途径的酶1-脱氧-D-木酮糖-5-磷酸合酶（DXS）转录水平的增加相关。中华猕猴桃萜烯合酶1（AcTPS1）被鉴定为八个串联重复基因阵列的一部分，并且在发育中的果实中仅观察到低水平的AcTPS1表达和萜烯生成。在本氏烟草叶片中瞬时过表达DXS可使AaTPS1的单萜合成提高100多倍，这表明DXS可能是调节猕猴桃中2-C-甲基-D-赤藓糖醇-4-磷酸底物通量的关键步骤。比较启动子分析确定了AaTPS1启动子中潜在的NAC（无顶端分生组织 [NAM]、拟南芥转录激活因子 [ATAF] 和杯状子叶 [CUC]）结构域转录因子和乙烯不敏感3样转录因子（TF）结合位点，并且在瞬时分析中，这两类TF的克隆成员都能够激活AaTPS1启动子。电泳迁移率变动分析表明，AaNAC2、AaNAC3和AaNAC4与AaTPS1启动子近端NAC结合位点的28 bp片段结合，但不与中华猕猴桃AcTPS1启动子结合，后者的NAC结合位点发生了突变。通过重新引入12 bp NAC核心结合基序的多个重复序列可以恢复激活。成熟的中华猕猴桃果实中缺乏NAC转录激活可解释该物种中AcTPS1转录本、蛋白质和单萜挥发物的低积累。这些结果表明NAC转录因子在控制成熟果实中的单萜生成和其他性状方面的重要性。
