Sasaki Takayuki, Tsuchiya Yoshiyuki, Ariyoshi Michiyo, Nakano Ryohei, Ushijima Koichiro, Kubo Yasutaka, Mori Izumi C, Higashiizumi Emi, Galis Ivan, Yamamoto Yoko
Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki, Okayama, 710-0046 Japan
Institute of Plant Science and Resources, Okayama University, Chuo 2-20-1, Kurashiki, Okayama, 710-0046 Japan.
Plant Cell Physiol. 2016 Nov;57(11):2367-2379. doi: 10.1093/pcp/pcw157. Epub 2016 Sep 11.
The aluminum-activated malate transporter (ALMT) family of proteins transports malate and/or inorganic anions across plant membranes. To demonstrate the possible role of ALMT genes in tomato fruit development, we focused on SlALMT4 and SlALMT5, the two major genes expressed during fruit development. Predicted proteins were classified into clade 2 of the family, many members of which localize to endomembranes. Tissue-specific gene expression was determined using transgenic tomato expressing the β-glucuronidase reporter gene controlled by their own promoters. Both the genes were expressed in vascular bundles connecting to developing seeds in fruit and in the embryo of mature seeds. Further, SlALMT5 was expressed in embryo in developing seeds in fruit. Subcellular localization of both proteins to the endoplasmic reticulum (ER) was established by transiently expressing the green fluorescent protein fusions in plant protoplasts. SlALMT5 probably localized to other endomembranes as well. Localization of SlALMT5 to the ER was also confirmed by immunoblot analysis. The transport function of both SlALMT proteins was investigated electrophysiologically in Xenopus oocytes. SlALMT5 transported malate and inorganic anions such as nitrate and chloride, but not citrate. SlALMT4 also transported malate, but the results were less consistent perhaps because it did not localize strongly to the plasma membrane. To elucidate the physiological role of SlALMT5 further, we overexpressed SlALMT5 in tomato. Compared with the wild type, overexpressors exhibited higher malate and citrate contents in mature seeds, but not in fruit. We conclude that the malate transport function of SlALMT5 expressed in developing fruit influences the organic acid contents in mature seeds.
铝激活苹果酸转运蛋白（ALMT）家族蛋白可跨植物膜转运苹果酸和/或无机阴离子。为了证明ALMT基因在番茄果实发育中的可能作用，我们重点研究了SlALMT4和SlALMT5，这是在果实发育过程中表达的两个主要基因。预测的蛋白质被归类为该家族的第2进化枝，其中许多成员定位于内膜。使用由其自身启动子控制的表达β-葡萄糖醛酸酶报告基因的转基因番茄来确定组织特异性基因表达。这两个基因在连接到果实中发育种子的维管束以及成熟种子的胚中均有表达。此外，SlALMT5在果实中发育种子的胚中表达。通过在植物原生质体中瞬时表达绿色荧光蛋白融合体，确定了这两种蛋白质在内质网（ER）中的亚细胞定位。SlALMT5可能也定位于其他内膜。免疫印迹分析也证实了SlALMT5定位于内质网。在非洲爪蟾卵母细胞中用电生理学方法研究了两种SlALMT蛋白的转运功能。SlALMT5转运苹果酸和无机阴离子，如硝酸盐和氯离子，但不转运柠檬酸。SlALMT4也转运苹果酸，但结果不太一致，可能是因为它没有强烈定位于质膜。为了进一步阐明SlALMT5的生理作用，我们在番茄中过表达了SlALMT5。与野生型相比，过表达植株在成熟种子中苹果酸和柠檬酸含量较高，但在果实中没有。我们得出结论，在发育中的果实中表达的SlALMT5的苹果酸转运功能影响成熟种子中的有机酸含量。
