Pereira Cláudia, Vieira Vanessa, Pissarra José, Pereira Susana
GreenUPorto-Sustainable Agrifood Production Research Centre/Inov4Agro, Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal.
Front Plant Sci. 2023 Jul 7;14:1085898. doi: 10.3389/fpls.2023.1085898. eCollection 2023.
The aspartic proteinase cardosin A is a vacuolar enzyme found to accumulate in protein storage and lytic vacuoles in the flowers and protein bodies in the seeds of the native plant cardoon. Cardosin A was first isolated several decades ago and has since been extensively characterized, both in terms of tissue distribution and enzyme biochemistry. In the native system, several roles have been attributed to cardosin A, such as reproduction, reserve mobilization, and membrane remodeling. To participate in such diverse events, cardosin A must accumulate and travel to different compartments within the cell: protein storage vacuoles, lytic vacuoles, and the cytoplasmic membrane (and eventually outside the cell). Several studies have approached the expression of cardosin A in and with promising results for the use of these systems to study of cardosin A trafficking. A poly-sorting mechanism has been uncovered for this protein, as two different vacuolar sorting determinants, mediating different vacuolar routes, have been described. The first is a conventional C-terminal domain, which delivers the protein to the vacuole via the Golgi, and the second is a more unconventional signal-the plant-specific insert (PSI)-that mediates a Golgi-independent route. The hypothesis that these two signals are activated according to cell needs and in organs with high metabolic activity is investigated here. An Arabidopsis line expressing cardosin A under an inducible promoter was used to understand the dynamics of cardosin A regarding vacuolar accumulation during seed germination events. Using antibodies against different regions of the protein and combining them with immunofluorescence and immunocytochemistry assays in different young seedling tissues, cardosin A was detected along the secretory pathway to the protein storage vacuole, often associated with the endoplasmic reticulum. More interestingly, upon treatment with the drug Brefeldin A, cardosin A was still detected in protein storage vacuoles, indicating that the intact protein can bypass the Golgi in this system, contrary to what was observed in . This study is a good starting point for further research involving the use of fluorescent fusions and exploring in more detail the relationship between cardosin A trafficking and plant development.
天冬氨酸蛋白酶卡多辛A是一种液泡酶，在当地植物刺菜蓟的花朵的蛋白质储存液泡和溶酶体以及种子的蛋白体中积累。卡多辛A在几十年前首次被分离出来，此后在组织分布和酶生物化学方面都得到了广泛的表征。在天然系统中，卡多辛A被赋予了多种作用，如繁殖、储备动员和膜重塑。为了参与这些不同的活动，卡多辛A必须积累并运输到细胞内的不同区室：蛋白质储存液泡、溶酶体和细胞质膜（最终到细胞外）。几项研究探讨了卡多辛A在[具体系统1]和[具体系统2]中的表达，对于利用这些系统研究卡多辛A的运输取得了有希望的结果。已经发现该蛋白存在一种多分选机制，因为已经描述了两种不同的液泡分选决定因素，介导不同的液泡途径。第一个是传统的C末端结构域，它通过高尔基体将蛋白质输送到液泡，第二个是更非传统的信号——植物特异性插入序列（PSI）——它介导一条不依赖高尔基体的途径。本文研究了这两种信号是否根据细胞需求并在具有高代谢活性的器官中被激活这一假设。利用一个在可诱导启动子控制下表达卡多辛A的拟南芥株系来了解种子萌发过程中卡多辛A在液泡积累方面的动态变化。使用针对该蛋白不同区域的抗体，并将它们与不同幼苗组织中的免疫荧光和免疫细胞化学分析相结合，在通向蛋白质储存液泡的分泌途径中检测到了卡多辛A，它通常与内质网相关。更有趣的是，在用布雷菲德菌素A处理后，在蛋白质储存液泡中仍检测到卡多辛A，这表明完整的蛋白质在该系统中可以绕过高尔基体，这与在[具体研究对象]中观察到的情况相反。这项研究是进一步开展涉及使用荧光融合以及更详细探索卡多辛A运输与植物发育之间关系的研究的良好起点。
