Introduction

In plants, malate, fumarate and citrate have central functions in cell metabolism. These organic acids are involved in metabolic pathways such as the synthesis and degradation of starch and sugars, in cellular respiration and in photosynthetic carbon fixation. A significant part of the carbon fixed during photosynthesis in plant cells is stored as malate and, in some plant species including Arabidopsis thaliana, also as fumarate (Chia et al., 2000). Additionally, citrate, malate and fumarate are involved in amino acid biosynthesis, the regulation of cellular pH (Hurth et al., 2005) and tolerance to heavy metal (Sasaki et al., 2004). In guard cells, malate is part of the molecular mechanisms regulating the opening and the closure of stomata (Roelfsema & Hedrich, 2005; Fernie & Martinoia, 2009; Araújo et al., 2011; Meyer et al., 2011).

In some plants, including A. thaliana, fumarate and malate are accumulated at similar levels, and together they form the major pool of C₄-organic acids in plant cells (Zell et al., 2010). Malate and fumarate are metabolically connected through fumarase enzymes, which reversibly converts malate into fumarate. Remarkably, in A. thaliana, two fumarase isoforms exist, the Fumarase 1 (FUM1, At2g47510) and the Fumarase 2 (FUM2, At5g50950). FUM1 is localised in the mitochondria and is part of the Krebs cycle (Heazlewood & Millar, 2005) while FUM2 is a cytosolic enzyme (Pracharoenwattana et al., 2010). The function of the FUM2 is associated with the accumulation of fumarate in mesophyll cells (Pracharoenwattana et al., 2010). Such an accumulation seems to be linked to the adaptation to low temperature and water restriction (Muller et al., 2011; Saunders et al., 2022). Recent reports using metabolic flux modelling propose that the conversion of malate into fumarate is a metabolic fail-safe to keep malate at levels in the cytosol (Saunders et al., 2022). Indeed, because of its central role in plant metabolism, cytosolic malate needs to be tightly regulated. Therefore, the reversible conversion of cytosolic malate into fumarate by FUM2 keeps cytosolic malate at metabolically compatible levels. Since malate and fumarate are mainly stored in the vacuole, their transport across the tonoplast regulates cytosolic homeostasis (Hurth et al., 2005; Medeiros et al., 2017). Being the principal store of malate and fumarate, the vacuole is part of the mechanisms regulating plant cell metabolism (Martinoia et al., 2007). The vacuolar concentration of these organic acids follows the circadian cycle, with malate and fumarate stored in the vacuole during daytime and remobilised at night for cellular respiration (Gibon et al., 2009). Instead, citrate, a major product of the Krebs cycle, is stored in the vacuole during the night and remobilised during the day and is a source of carbon skeletons for light-dependent nitrate assimilation (Tcherkez et al., 2012; Cheung et al., 2014; Winter & Smith, 2022).

Different transport systems mediate the fluxes of metabolites across the tonoplast. The first vacuolar organic acid transporter identified, AttDT, was associated with the control of pH homeostasis and in storing/remobilising dicarboxylates in the vacuole (Hurth et al., 2005). AttDT catalyses the exchange of vacuolar malate with cytosolic citrate (Frei et al., 2018). In the leaves, Attdt knock-out plants present reduced malate and fumarate content compared with wild-type (WT), but increased citrate (Medeiros et al., 2017; Frei et al., 2018).

The ion channels of the Aluminium-Activated Malate Transporter (ALMT) family are able to mediate malate and fumarate transport. The ALMTs are membrane proteins present only in plants and, in Arabidopsis, the 14 members are divided in three clades (Kovermann et al., 2007). The members of the ALMT family are involved in a variety of functions, including tolerance to aluminium in the soil, mineral nutrition, regulation of fruit acidity (Sharma et al., 2016; Li et al., 2020), ion homeostasis and stomatal aperture regulation (Meyer et al., 2011; De Angeli et al., 2013; Eisenach et al., 2017; Sasaki et al., 2022). Clade I is formed by six ALMTs localised at the plasma membrane, and it includes the first identified AtALMT, AtALMT1 (Sasaki et al., 2004). In the roots, AtALMT1 mediates malate efflux to chelate aluminium in the soil, conferring Al³⁺ tolerance. Clade III is formed by four members and includes the plasma membrane ion channel AtALMT12 that is involved in the regulation of stomatal aperture (Meyer et al., 2010; Sasaki et al., 2022). Finally, Clade II presents three members: AtALMT4, AtALMT6 and AtALMT9, localised at the vacuolar membrane and one, AtALMT3, localised at the plasma membrane (Meyer et al., 2011; De Angeli et al., 2013; Baetz et al., 2016; Eisenach et al., 2017; Maruyama et al., 2019). AtALMT9 is expressed in the roots and the shoots (Baetz et al., 2016). In the leaves, it can mediate malate influx in the vacuole of mesophyll cells, and in guard cells, it most likely mediates chloride uptake in the vacuole during stomatal opening (De Angeli et al., 2013). AtALMT4 mediates the efflux of malate from the vacuole and regulates abscisic acid (ABA)-induced stomatal closure (Eisenach et al., 2017). Finally, AtALMT6 is specifically expressed in guard cells and mediates both malate influx and efflux from the vacuole and is activated by cytosolic Ca²⁺ (Meyer et al., 2011; Ye et al., 2021). However, even if electrophysiological approaches demonstrated that these vacuolar ALMTs are able to mediate malate and fumarate transport across the tonoplast, no significant difference in the content of these organic acids could be detected in the respective Arabidopsis knock-out mutants (Baetz et al., 2016; Eisenach et al., 2017). This keep the issue of their role in vivo as tonoplastic malate transport systems an open question.

Notably, AtALMT5 has been the forgotten member of Clade II, and almost no information about it is available. Indeed, its functional characteristics as well as its role in vivo are still unknown. Therefore, in this study, we decided to tackle the issue of the function of AtALMT5 in Arabidopsis. Our findings show that AtALMT5 is localised in the vacuolar membrane and is specifically expressed in the shoots. We found that AtALMT5 preferentially mediates fumarate currents in a physiological range of transmembrane potentials and that vacuoles from almt5 mutants have reduced fumarate currents. We demonstrated that in vivo AtALMT5 mediates fumarate accumulation in the leaves and that in almt5 knock-out plants, the malate/fumarate ratio is modified. Overall, our data identify AtALMT5 as a transporter-mediating fumarate transport across the vacuolar membrane of Arabidopsis. In the leaves, AtALMT5 regulates the accumulation of organic acids produced by photosynthesis.

中文翻译：

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AtALMT5 介导液泡富马酸盐输入并调节拟南芥中的苹果酸/富马酸盐平衡

 介绍

在植物中，苹果酸盐、富马酸盐和柠檬酸盐在细胞代谢中具有核心功能。这些有机酸参与代谢途径，例如淀粉和糖的合成和降解、细胞呼吸和光合碳固定。植物细胞光合作用过程中固定的碳的很大一部分以苹果酸盐的形式储存，并且在包括拟南芥在内的一些植物物种中，也以富马酸盐的形式储存（Chia等 人，2000 年）。此外，柠檬酸盐、苹果酸盐和富马酸盐参与氨基酸生物合成、细胞 pH 调节（Hurth等 人，2005 年）和对重金属的耐受性（Sasaki 等 人，2004 年）。在保卫细胞中，苹果酸是调节气孔打开和关闭的分子机制的一部分（Roelfsema & Hedrich，2005;Fernie & Martinoia， 2009;Araújo et al.， 2011;Meyer et al.， 2011）。

在一些植物中，包括 A. thaliana，富马酸盐和苹果酸盐以相似的水平积累，它们一起在植物细胞中形成 C₄-有机酸的主要库（Zell等 人，2010 年）。苹果酸和富马酸盐通过富马酸盐酶代谢连接，富马酸盐酶可逆地将苹果酸转化为富马酸盐。值得注意的是，在 A. thaliana 中，存在两种富马酶亚型，即富马酶 1 （FUM1， 在2g47510） 和富马酶 2 （FUM2， 在5g50950）。FUM1位于线粒体中，是克雷布斯循环的一部分（Heazlewood & Millar（2005），而FUM2是一种胞质酶（Pracharoenwattana等 人，2010）。FUM2 的功能与富马酸盐在叶肉细胞中的积累有关（Pracharoenwattana等 人，2010 年）。这种积累似乎与对低温和水限制的适应有关（Muller et al.， 2011;Saunders et al.， 2022）。最近使用代谢通量模型的报告表明，苹果酸转化为富马酸盐是一种代谢故障安全，可将苹果酸保持在胞质溶胶中的水平（Saunders等 人，2022 年）。事实上，由于其在植物代谢中的核心作用，胞质苹果酸需要受到严格调节。因此，FUM2 将胞质苹果酸盐可逆地转化为富马酸盐，使胞质苹果酸盐保持在代谢相容水平。由于苹果酸和富马酸盐主要储存在液泡中，因此它们穿过核质体的运输调节胞质稳态（Hurth等人 ，2005 年;Medeiros et al.， 2017）。 作为苹果酸和富马酸盐的主要储存库，液泡是调节植物细胞代谢机制的一部分（Martinoia等 人，2007 年）。这些有机酸的液泡浓度遵循昼夜节律周期，苹果酸和富马酸盐在白天储存在液泡中，并在夜间重新动员以进行细胞呼吸（Gibon et al.， 2009）。相反，柠檬酸盐是克雷布斯循环的主要产物，在夜间储存在液泡中，并在白天重新动员，并且是光依赖性硝酸盐同化的碳骨架来源（Tcherkez等 人，2012 年;Cheung等 人，2014 年;Winter & Smith，2022 年）。

不同的运输系统介导代谢物穿过 Tonoplast 的通量。第一个发现的液泡有机酸转运蛋白 AttDT 与 pH 稳态的控制以及在液泡中储存/再动员二羧酸盐有关（Hurth等 人，2005 年）。AttDT 催化液泡苹果酸与胞质柠檬酸盐的交换（Frei等 人，2018 年）。在叶子中，与野生型 （WT） 相比，Attdt 敲除植物的苹果酸盐和富马酸盐含量降低，但柠檬酸盐含量增加（Medeiros等 人，2017 年;Frei et al.， 2018）。

铝活化苹果酸转运蛋白 （ALMT） 家族的离子通道能够介导苹果酸和富马酸盐转运。ALMT 是仅存在于植物中的膜蛋白，在拟南芥中，14 个成员分为三个分支（Kovermann等 人，2007 年）。ALMT 家族的成员参与多种功能，包括对土壤中铝的耐受性、矿物质营养、水果酸度的调节（Sharma 等 人，2016 年;Li等人 ，2020 年）、离子稳态和气孔孔径调节（Meyer等人 ，2011 年;De Angeli 等 人，2013 年;Eisenach等 人，2017 年;Sasaki et al.， 2022）。进化枝 I 由定位于质膜的 6 个 ALMT 形成，它包括第一个在 ALMT、ALMT1 发现的 ALMT（Sasaki 等 人，2004 年）。在根中，AtALMT1 介导苹果酸外排以螯合土壤中的铝，赋予 Al³⁺ 耐受性。进化枝 III 由四个成员形成，包括参与气孔孔径调节的质膜离子通道 ALMT12（Meyer等人 ，2010 年;Sasaki et al.， 2022）。最后，分支 II 呈现三个成员：在ALMT4、ALMT6 和ALMT9 处，位于液泡膜，一个在ALMT3 处，位于质膜（Meyer等 人，2011 年;De Angeli 等 人，2013 年;Baetz et al.， 2016;Eisenach 等 人。，2017 年;Maruyama等 人，2019 年）。在ALMT9 在根和芽中表达（Baetz等人 ，2016 年）。在叶子中，它可以介导叶肉细胞液泡中的苹果酸内流，而在保卫细胞中，它很可能在气孔打开期间介导液泡中氯的摄取（De Angeli等 人，2013）。在ALMT4 介导苹果酸从液泡中流出并调节脱落酸 （ABA） 诱导的气孔关闭 （Eisenach et al.， 2017）。最后，AtALMT6 在保卫细胞中特异性表达，介导苹果酸内流和液泡外流，并被胞质 Ca²⁺ 激活（Meyer等人 ，2011 年;Ye et al.， 2021）。然而，即使电生理学方法证明这些液泡 ALMT 能够介导苹果酸盐和富马酸盐跨托质体的运输，在相应的拟南芥敲除突变体中也无法检测到这些有机酸含量的显着差异（Baetz等人 ，2016 年;Eisenach et al.， 2017）。这使得它们在体内作为钙质塑性苹果酸盐运输系统的作用问题成为一个悬而未决的问题。

值得注意的是，AtALMT5 一直是 Clade II 中被遗忘的成员，几乎没有关于它的信息。事实上，它的功能特性及其在体内的作用仍然未知。因此，在这项研究中，我们决定解决拟南芥中 AtALMT5 的功能问题。我们的研究结果表明，AtALMT5 位于液泡膜中，并在芽中特异性表达。我们发现 AtALMT5 优先介导跨膜电位生理范围内的富马酸盐电流，并且来自 ALMT5 突变体的液泡降低了富马酸盐电流。我们证明体内 AtALMT5 介导富马酸盐在叶子中的积累，并且在 ALMT5 敲除植物中，苹果酸/富马酸盐比率被改变。总体而言，我们的数据将 AtALMT5 确定为转运蛋白介导的富马酸盐转运穿过拟南芥液泡膜。在叶子中，AtALMT5 调节光合作用产生的有机酸的积累。