Australia-China Joint Centre for Wheat Improvement, Western Australian State Agriculture Biotechnology Centre, School of Veterinary and Life Sciences, Murdoch University, Perth, WA, 6150, Australia.
The Key Laboratory for Quality Improvement of Agricultural Products of Zhejiang Province, School of Agriculture and Food Science, Zhejiang A&F University, Linan, Zhejiang, 311300, Hangzhou, China.
BMC Plant Biol. 2020 Jan 29;20(1):45. doi: 10.1186/s12870-020-2259-z.
Wheat grain avenin-like proteins (ALPs) belong to a recently discovered class of wheat grain storage protein. ALPs in wheat grains not only have beneficial effects on dough quality but also display antifungal activities, which is a novel observation for wheat storage proteins. Previous studies have shown that ALPs are likely present in the albumin/globulin fractions of total protein extract from wheat flour. However, the accumulation characteristics of these ALPs in the mature wheat grain remains unknown.
In the present study, a total of 13 ALPs homologs were isolated and characterized in the albumin/globulin fractions of the wheat protein extract. A combination of multiple techniques including RP-HPLC, SDS-PAGE, MALDI-TOF and peptide sequencing were used for accurate separation and identification of individual ALP homolog. The C-terminal TaALP-by-4AL/7DS, TaALP-by-4AL/7AS/7DS, TaALP-bx/4AL/7AS/7DS, TaALP-ay-7DS, TaALP-ay-4AL, TaALP-ax-4AL, TaALP-ax-7AS, and TaALP-ax-7DS, were separated as individual protein bands from wheat flour for the first time. These unique ALPs peptides were mapped to the latest wheat genome assembly in the IWGSC database. The characteristic defence related proteins present in albumin and globulin fractions, such as protein disulfide-isomerase (PDI), grain softness protein (GSP), alpha-amylase inhibitors (AAIs) and endogenous alpha-amylase/subtilisin inhibitor were also found to co-segregate with these identified ALPs, avenin-3 and α-gliadins. The molecular weight range and the electrophoresis segregation properties of ALPs were characterised in comparison with the proteins containing the tryp_alpha_amyl domain (PF00234) and the gliadin domain (PF13016), which play a role in plant immunity and grain quality. We examined the phylogenetic relationships of the AAIs, GSP, avenin-3, α-gliadins and ALPs, based on the alignment of their functional domains. MALDI-TOF profiling indicated the occurrence of certain post-translations modifications (PTMs) in some ALP subunits.
We reported for the first time the complete profiling of ALPs present in the albumin/globulin fractions of wheat grain protein extracts. We concluded that majority of the ALPs homologs are expressed in wheat grains. We found clear evidence of PTMs in several ALPs peptides. The identification of both gliadin domain (PF13016) and Tryp_alpha_amyl domain (PF00234) in the mature forms of ALPs highlighted the multiple functional properties of ALPs in grain quality and disease resistance.
小麦醇溶蛋白样蛋白（ALPs）属于最近发现的一类小麦贮藏蛋白。小麦籽粒中的 ALPs 不仅对面团质量有有益的影响，而且还具有抗真菌活性，这是对小麦贮藏蛋白的新观察。先前的研究表明，ALPs 可能存在于小麦粉中总蛋白提取物的白蛋白/球蛋白部分。然而，这些 ALPs 在成熟小麦籽粒中的积累特征尚不清楚。
在本研究中，从小麦蛋白提取物的白蛋白/球蛋白部分共分离和鉴定了 13 种 ALPs 同源物。采用反相高效液相色谱法（RP-HPLC）、SDS-PAGE、MALDI-TOF 和肽测序等多种技术相结合，对单个 ALP 同源物进行了准确的分离和鉴定。首次从小麦粉中分离出 TaALP-by-4AL/7DS、TaALP-by-4AL/7AS/7DS、TaALP-bx/4AL/7AS/7DS、TaALP-ay-7DS、TaALP-ay-4AL、TaALP-ax-4AL、TaALP-ax-7AS 和 TaALP-ax-7DS 等 C 端 TaALP-by-4AL/7DS、TaALP-by-4AL/7AS/7DS、TaALP-bx/4AL/7AS/7DS、TaALP-ay-7DS、TaALP-ay-4AL、TaALP-ax-4AL、TaALP-ax-7AS 和 TaALP-ax-7DS 作为单个蛋白质条带。这些独特的 ALP 肽被映射到 IWGSC 数据库中最新的小麦基因组组装上。在白蛋白和球蛋白部分还发现了与这些鉴定的 ALPs、醇溶蛋白 3 和 α-麦谷蛋白共分离的特征防御相关蛋白，如蛋白二硫键异构酶（PDI）、谷物柔软蛋白（GSP）、α-淀粉酶抑制剂（AAI）和内源性α-淀粉酶/枯草杆菌蛋白酶抑制剂。比较含有 trypsin_alpha_amylase 结构域（PF00234）和 gliadin 结构域（PF13016）的蛋白质，研究了 ALPs、醇溶蛋白 3、α-麦谷蛋白和 AAls 的分子质量范围和电泳分离特性，这两种结构域在植物免疫和谷物质量中起作用。我们根据其功能结构域的比对，研究了 AAls、GSP、醇溶蛋白 3、α-麦谷蛋白和 ALPs 的系统发育关系。MALDI-TOF 分析表明，一些 ALP 亚基存在某些翻译后修饰（PTMs）。
我们首次全面描述了小麦籽粒蛋白提取物白蛋白/球蛋白部分中存在的 ALPs。我们得出结论，大多数 ALPs 同源物在小麦籽粒中表达。我们在几个 ALP 肽中发现了明显的 PTMs 证据。在 ALPs 的成熟形式中发现了麦醇溶蛋白结构域（PF13016）和 trypsin_alpha_amylase 结构域（PF00234），这突出了 ALPs 在谷物质量和抗病性中的多种功能特性。
