Ceballos-Laita Laura, Gutierrez-Carbonell Elain, Lattanzio Giuseppe, Vázquez Saul, Contreras-Moreira Bruno, Abadía Anunciación, Abadía Javier, López-Millán Ana-Flor
Plant Stress Physiology Group, Department of Plant Nutrition, Aula Dei Experimental Station, Consejo Superior de Investigaciones Científicas Zaragoza, Spain.
Laboratory of Computational and Structural Biology, Aula Dei Experimental Station, Consejo Superior de Investigaciones Científicas Zaragoza, Spain ; Fundación ARAID Zaragoza, Spain.
Front Plant Sci. 2015 Mar 18;6:145. doi: 10.3389/fpls.2015.00145. eCollection 2015.
The fluid collected by direct leaf centrifugation has been used to study the proteome of the sugar beet apoplastic fluid as well as the changes induced by Fe deficiency and Fe resupply to Fe-deficient plants in the protein profile. Plants were grown in Fe-sufficient and Fe-deficient conditions, and Fe resupply was carried out with 45 μM Fe(III)-EDTA for 24 h. Protein extracts of leaf apoplastic fluid were analyzed by two-dimensional isoelectric focusing-SDS-PAGE electrophoresis. Gel image analysis revealed 203 consistent spots, and proteins in 81% of them (164) were identified by nLC-MS/MS using a custom made reference repository of beet protein sequences. When redundant UniProt entries were deleted, a non-redundant leaf apoplastic proteome consisting of 109 proteins was obtained. TargetP and SecretomeP algorithms predicted that 63% of them were secretory proteins. Functional classification of the non-redundant proteins indicated that stress and defense, protein metabolism, cell wall and C metabolism accounted for approximately 75% of the identified proteome. The effects of Fe-deficiency on the leaf apoplast proteome were limited, with only five spots (2.5%) changing in relative abundance, thus suggesting that protein homeostasis in the leaf apoplast fluid is well-maintained upon Fe shortage. The identification of three chitinase isoforms among proteins increasing in relative abundance with Fe-deficiency suggests that one of the few effects of Fe deficiency in the leaf apoplast proteome includes cell wall modifications. Iron resupply to Fe deficient plants changed the relative abundance of 16 spots when compared to either Fe-sufficient or Fe-deficient samples. Proteins identified in these spots can be broadly classified as those responding to Fe-resupply, which included defense and cell wall related proteins, and non-responsive, which are mainly protein metabolism related proteins and whose changes in relative abundance followed the same trend as with Fe-deficiency.
通过直接叶片离心收集的液体已用于研究甜菜质外体液体的蛋白质组，以及缺铁和向缺铁植物重新供应铁时蛋白质谱的变化。植物在铁充足和缺铁条件下生长，并用45 μM Fe(III)-EDTA进行24小时的铁再供应。通过二维等电聚焦-SDS-PAGE电泳分析叶质外体液体的蛋白质提取物。凝胶图像分析显示了203个一致的斑点，其中81%（164个）的蛋白质通过使用定制的甜菜蛋白质序列参考库的nLC-MS/MS进行了鉴定。删除冗余的UniProt条目后，获得了由109种蛋白质组成的非冗余叶质外体蛋白质组。TargetP和SecretomeP算法预测其中63%是分泌蛋白。对非冗余蛋白质的功能分类表明，应激和防御、蛋白质代谢、细胞壁和碳代谢约占已鉴定蛋白质组的75%。缺铁对叶质外体蛋白质组的影响有限，相对丰度仅5个斑点（2.5%）发生变化，这表明在缺铁时叶质外体液体中的蛋白质稳态得到了很好的维持。在缺铁时相对丰度增加的蛋白质中鉴定出三种几丁质酶同工型，这表明缺铁在叶质外体蛋白质组中的少数影响之一包括细胞壁修饰。与铁充足或缺铁样品相比，向缺铁植物重新供应铁改变了16个斑点的相对丰度。在这些斑点中鉴定出的蛋白质大致可分为对铁再供应有反应的蛋白质，包括防御和细胞壁相关蛋白质，以及无反应的蛋白质，主要是蛋白质代谢相关蛋白质，其相对丰度的变化与缺铁时的趋势相同。
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