Lara Juan A, Burciaga-Monge Alma, Chávez Angel, Revés Marc, Lavilla Rodolfo, Arró Monserrat, Boronat Albert, Altabella Teresa, Ferrer Albert
Plant Metabolism and Metabolic Engineering Program, Centre for Research in Agricultural Genomics (CRAG) (CSIC-IRTA-UAB-UB), Campus Autonomous University of Barcelona, Cerdanyola del Vallès, Spain.
Laboratory of Medicinal Chemistry, Institute of Biomedicine University of Barcelona, Faculty of Pharmacy and Food Sciences, University of Barcelona, Barcelona, Spain.
Front Plant Sci. 2018 May 8;9:588. doi: 10.3389/fpls.2018.00588. eCollection 2018.
Steryl esters (SEs) serve as a storage pool of sterols that helps to maintain proper levels of free sterols (FSs) in cell membranes throughout plant growth and development, and participates in the recycling of FSs and fatty acids released from cell membranes in aging tissues. SEs are synthesized by sterol acyltransferases, a family of enzymes that catalyze the transfer of fatty acil groups to the hydroxyl group at C-3 position of the sterol backbone. Sterol acyltransferases are categorized into acyl-CoA:sterol acyltransferases (ASAT) and phospholipid:sterol acyltransferases (PSAT) depending on whether the fatty acyl donor substrate is a long-chain acyl-CoA or a phospolipid. Until now, only Arabidopsis ASAT and PSAT enzymes (AtASAT1 and AtPSAT1) have been cloned and characterized in plants. Here we report the identification, cloning, and functional characterization of the tomato ( cv. Micro-Tom) orthologs. SlPSAT1 and SlASAT1 were able to restore SE to wild type levels in the Arabidopsis and knock-out mutants, respectively. Expression of SlPSAT1 in the background also prevented the toxicity caused by an external supply of mevalonate and the early senescence phenotype observed in detached leaves of this mutant, whereas expression of SlASAT1 in the mutant revealed a clear substrate preference of the tomato enzyme for the sterol precursors cycloartenol and 24-methylene cycloartanol. Subcellular localization studies using fluorescently tagged SlPSAT1 and SlASAT1 proteins revealed that SlPSAT1 localize in cytoplasmic lipid droplets (LDs) while, in contrast to the endoplasmic reticulum (ER) localization of AtASAT1, SlASAT1 resides in the plasma membrane (PM). The possibility that PM-localized SlASAT1 may act catalytically on their sterol substrates, which are presumably embedded in the ER membrane, is discussed. The widespread expression of and genes in different tomato organs together with their moderate transcriptional response to several stresses suggests a dual role of SlPSAT1 and SlASAT1 in tomato plant and fruit development and the adaptive responses to stress. Overall, this study contributes to enlarge the current knowledge on plant sterol acyltransferases and set the basis for further studies aimed at understanding the role of SE metabolism in tomato plant growth and development.
甾醇酯（SEs）作为甾醇的储存库，有助于在植物整个生长发育过程中维持细胞膜中游离甾醇（FSs）的适当水平，并参与衰老组织中从细胞膜释放的FSs和脂肪酸的循环利用。SEs由甾醇酰基转移酶合成，该酶家族催化脂肪酰基转移至甾醇骨架C-3位的羟基上。甾醇酰基转移酶根据脂肪酰基供体底物是长链酰基辅酶A还是磷脂，分为酰基辅酶A：甾醇酰基转移酶（ASAT）和磷脂：甾醇酰基转移酶（PSAT）。到目前为止，在植物中仅克隆和鉴定了拟南芥ASAT和PSAT酶（AtASAT1和AtPSAT1）。在此，我们报道番茄（品种Micro-Tom）直系同源物的鉴定、克隆及功能特性。SlPSAT1和SlASAT1分别能够使拟南芥中SE恢复到野生型水平以及敲除突变体中SE恢复到野生型水平。在该突变体背景下SlPSAT1的表达还能防止外源供应甲羟戊酸所引起的毒性以及该突变体离体叶片中观察到的早期衰老表型，而在该突变体中SlASAT1的表达揭示了番茄酶对甾醇前体环阿屯醇和24-亚甲基环阿屯醇有明显的底物偏好。使用荧光标记的SlPSAT1和SlASAT1蛋白进行的亚细胞定位研究表明，SlPSAT1定位于细胞质脂滴（LDs），而与AtASAT1定位于内质网（ER）相反，SlASAT1存在于质膜（PM）中。讨论了质膜定位的SlASAT1可能对其推测嵌入内质网膜中的甾醇底物起催化作用的可能性。SlPSAT1和SlASAT1基因在不同番茄器官中的广泛表达以及它们对几种胁迫的适度转录反应表明，SlPSAT1和SlASAT1在番茄植株和果实发育以及对胁迫的适应性反应中具有双重作用。总体而言，本研究有助于扩展当前关于植物甾醇酰基转移酶的知识，并为进一步研究奠定基础，旨在了解SE代谢在番茄植株生长发育中的作用。
