Tanner Gregory J, Colgrave Michelle L, Blundell Malcolm J, Howitt Crispin A, Bacic Antony
School of Biosciences, University of Melbourne, Melbourne, VIC, Australia.
Commonwealth Scientific and Industrial Research Organisation Agriculture and Food, St Lucia, QLD, Australia.
Front Plant Sci. 2019 May 16;10:649. doi: 10.3389/fpls.2019.00649. eCollection 2019.
The temporal pattern of accumulation of hordein storage proteins in developing barley grains was studied by enzyme-linked immunosorbent assay (ELISA), western blot and liquid chromatography tandem mass spectrometry (LC-MS/MS). Hordein accumulation was compared to the pattern seen for two abundant control proteins, serpin Z4 (an early accumulator) and lipid transferase protein (LTP1, a late accumulator). Hordeins were detected from 6 days post-anthesis (DPA) and peaked at 30 DPA. Changes in fresh weight indicate that desiccation begins at 20 DPA and by 37 DPA fresh weight had decreased by 35%. ELISA analysis of hordein content, expressed on a protein basis, increased to a maximum at 30 DPA followed by a 17% decrease by 37 DPA. The accumulation of 39 tryptic and 29 chymotryptic hordein peptides representing all classes of hordein was studied by LC-MS/MS. Most peptides increased to a maximum at 30 DPA, and either remained at the maximum or did not decrease significantly. Only five tryptic peptides, members of the related B1- and γ1-hordeins decreased significantly by 21-51% at 37 DPA. Thus, the concentration of some specific peptides was reduced while remaining members of the same family were not affected. The N-terminal signal region was removed by proteolysis during co-translation. In addition to a suite of previously characterized hordeins, two novel barley B-hordein isoforms mapping to wheat low molecular weight glutenins (LMW-GS-like B-hordeins), and two avenin-like proteins (ALPs) sharing homology with wheat ALPs, were identified. These identified isoforms have not previously been mapped in the barley genome. Cereal storage proteins provide significant nutritional content for human consumption and seed germination. In barley, the bulk of the storage proteins comprise the hordein family and the final hordein concentration affects the quality of baked and brewed products. It is therefore important to study the accumulation of hordeins as this knowledge may assist plant breeding for improved health outcomes (by minimizing triggering of detrimental immune responses), nutrition and food processing properties.
通过酶联免疫吸附测定（ELISA）、蛋白质免疫印迹法和液相色谱串联质谱法（LC-MS/MS）研究了发育中的大麦籽粒中醇溶蛋白储存蛋白的积累时间模式。将醇溶蛋白的积累模式与两种丰富的对照蛋白（丝氨酸蛋白酶抑制剂Z4（一种早期积累蛋白）和脂质转移蛋白（LTP1，一种晚期积累蛋白））的积累模式进行了比较。在开花后6天（DPA）检测到醇溶蛋白，并在30 DPA时达到峰值。鲜重变化表明，干燥在20 DPA开始，到37 DPA时鲜重下降了35%。以蛋白质为基础对醇溶蛋白含量进行的ELISA分析显示，在30 DPA时增加到最大值，随后到37 DPA时下降了17%。通过LC-MS/MS研究了代表所有醇溶蛋白类别的39种胰蛋白酶水解和29种糜蛋白酶水解醇溶蛋白肽段的积累情况。大多数肽段在30 DPA时增加到最大值，要么保持在最大值，要么没有显著下降。只有5种胰蛋白酶肽段，即相关的B1-和γ1-醇溶蛋白成员，在37 DPA时显著下降了21%-51%。因此，一些特定肽段的浓度降低，而同一家族的其他成员不受影响。N端信号区域在共翻译过程中通过蛋白水解作用被去除。除了一组先前已鉴定的醇溶蛋白外，还鉴定出了两种新的大麦B-醇溶蛋白亚型，它们与小麦低分子量谷蛋白（LMW-GS样B-醇溶蛋白）定位相同，以及两种与小麦醇溶蛋白样蛋白（ALP）具有同源性的燕麦醇溶蛋白样蛋白（ALP）。这些已鉴定的亚型以前未在大麦基因组中定位。谷物储存蛋白为人类消费和种子萌发提供了重要的营养成分。在大麦中，大部分储存蛋白由醇溶蛋白家族组成，最终的醇溶蛋白浓度会影响烘焙和酿造产品的质量。因此，研究醇溶蛋白的积累很重要，因为这些知识可能有助于植物育种，以改善健康结果（通过尽量减少有害免疫反应）、营养和食品加工特性。
