The Arabidopsis vacuolar malate channel is a member of the ALMT family
The Arabidopsis vacuolar malate channel is a member of the ALMT family
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DOI：
10.1111/j.1365-313x.2007.03367.x
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发表时间：
2007-12-01
期刊：
影响因子：
7.2
通讯作者：
Martinoia, Enrico
中科院分区：
文献类型：
--
作者：
Kovermann, Peter;Meyer, Stefan;Martinoia, Enrico
在植物中，苹果酸是一种中心代谢物，具有多种功能。液泡苹果酸可能达到非常高的浓度并快速波动，而胞质苹果酸则保持在恒定水平，从而实现最佳代谢。最近，鉴定出一种液泡苹果酸转运蛋白（拟南芥液泡膜二羧酸转运蛋白，AttDT），它与充分表征的液泡苹果酸通道不相符。因此，我们假设铝激活苹果酸转运蛋白（ALMT）基因家族的成员可以编码液泡苹果酸通道。使用 GFP 融合构建体，我们可以证明 AtALMT9 (A. thaliana ALMT9) 靶向液泡。启动子-GUS 融合构建体证明该基因在所有器官中表达，但具有细胞类型特异性，因为叶子中的 GUS 活性几乎只在叶肉细胞中检测到。 Atalmt9 T-DNA 插入突变体的膜片钳分析显示液泡苹果酸通道活性强烈降低。为了在功能上表征 AtALMT9 作为苹果酸通道的特征，我们在烟草和卵母细胞中异源表达该基因。 AtALMT9-GFP 在本塞姆氏烟草叶片中的过度表达强烈增强了叶肉液泡膜上的苹果酸电流密度。 AtALMT9 在爪蟾卵母细胞中的功能性表达诱导阴离子电流，这与内源卵母细胞电流明显区分。我们的结果表明 AtALMT9 是一个液泡苹果酸通道。 AtALMT9 的缺失突变体仅表现出叶肉原生质体中苹果酸含量略有降低，并且没有可见的表型，表明 AttDT 和残留的苹果酸通道活性足以维持调节胞质苹果酸稳态所需的转运活性。
In plants, malate is a central metabolite and fulfills a large number of functions. Vacuolar malate may reach very high concentrations and fluctuate rapidly, whereas cytosolic malate is kept at a constant level allowing optimal metabolism. Recently, a vacuolar malate transporter (Arabidopsis thaliana tonoplast dicarboxylate transporter, AttDT) was identified that did not correspond to the well-characterized vacuolar malate channel. We therefore hypothesized that a member of the aluminum-activated malate transporter (ALMT) gene family could code for a vacuolar malate channel. Using GFP fusion constructs, we could show that AtALMT9 (A. thaliana ALMT9) is targeted to the vacuole. Promoter-GUS fusion constructs demonstrated that this gene is expressed in all organs, but is cell-type specific as GUS activity in leaves was detected nearly exclusively in mesophyll cells. Patch-clamp analysis of an Atalmt9 T-DNA insertion mutant exhibited strongly reduced vacuolar malate channel activity. In order to functionally characterize AtALMT9 as a malate channel, we heterologously expressed this gene in tobacco and in oocytes. Overexpression of AtALMT9-GFP in Nicotiana benthamiana leaves strongly enhanced the malate current densities across the mesophyll tonoplasts. Functional expression of AtALMT9 in Xenopus oocytes induced anion currents, which were clearly distinguishable from endogenous oocyte currents. Our results demonstrate that AtALMT9 is a vacuolar malate channel. Deletion mutants for AtALMT9 exhibit only slightly reduced malate content in mesophyll protoplasts and no visible phenotype, indicating that AttDT and the residual malate channel activity are sufficient to sustain the transport activity necessary to regulate the cytosolic malate homeostasis.