Biopolymers Interactions Assemblies Research Unit, French National Institute for Agriculture, Food and Environment, Nantes, France.
Independent Researcher, Bénéjacq, France.
PLoS One. 2020 Sep 29;15(9):e0225293. doi: 10.1371/journal.pone.0225293. eCollection 2020.
Grain hardness is an important quality trait of cereal crops. In wheat, it is mainly determined by the Hardness locus that harbors genes encoding puroindoline A (PINA) and puroindoline B (PINB). Any deletion or mutation of these genes leading to the absence of PINA or to single amino acid changes in PINB leads to hard endosperms. Although it is generally acknowledged that hardness is controlled by adhesion strength between the protein matrix and starch granules, the physicochemical mechanisms connecting puroindolines and the starch-protein interactions are unknown as of this time. To explore these mechanisms, we focused on PINA. The overexpression in a hard wheat cultivar (cv. Courtot with the Pina-D1a and Pinb-D1d alleles) decreased grain hardness in a dose-related effect, suggesting an interactive process. When PINA was added to gliadins in solution, large aggregates of up to 13 μm in diameter were formed. Turbidimetry measurements showed that the PINA-gliadin interaction displayed a high cooperativity that increased with a decrease in pH from neutral to acid (pH 4) media, mimicking the pH change during endosperm development. No turbidity was observed in the presence of isolated α- and γ-gliadins, but non-cooperative interactions of PINA with these proteins could be confirmed by surface plasmon resonance. A significant higher interaction of PINA with γ-gliadins than with α-gliadins was observed. Similar binding behavior was observed with a recombinant repeated polypeptide that mimics the repeat domain of gliadins, i.e., (Pro-Gln-Gln-Pro-Tyr)8. Taken together, these results suggest that the interaction of PINA with a monomeric gliadin creates a nucleation point leading to the aggregation of other gliadins, a phenomenon that could prevent further interaction of the storage prolamins with starch granules. Consequently, the role of puroindoline-prolamin interactions on grain hardness should be addressed on the basis of previous observations that highlight the similar subcellular routing of storage prolamins and puroindolines.
谷物硬度是谷类作物的一个重要品质性状。在小麦中，它主要由硬度基因座决定，该基因座含有编码多聚谷氨酰胺 A（PINA）和多聚谷氨酰胺 B（PINB）的基因。这些基因的任何缺失或突变导致 PINA 缺失或 PINB 中的单个氨基酸变化都会导致硬质胚乳。尽管普遍认为硬度受蛋白质基质与淀粉颗粒之间的粘附强度控制，但目前尚不清楚多聚谷氨酰胺与淀粉-蛋白质相互作用之间的物理化学机制。为了探索这些机制，我们专注于 PINA。在硬粒小麦品种（Courtot 品种，具有 Pina-D1a 和 Pinb-D1d 等位基因）中的过表达以剂量相关的方式降低了籽粒硬度，表明存在相互作用过程。当 PINA 被添加到溶液中的麦醇溶蛋白中时，形成了高达 13 μm 直径的大聚集体。浊度测量表明，PINA-麦醇溶蛋白相互作用表现出高协同性，随着 pH 值从中性降低到酸性（pH4）介质而增加，模拟了胚乳发育过程中的 pH 变化。在存在分离的α-和γ-麦醇溶蛋白的情况下未观察到浊度，但可以通过表面等离子体共振证实 PINA 与这些蛋白质的非协同相互作用。观察到 PINA 与 γ-麦醇溶蛋白的相互作用明显高于与 α-麦醇溶蛋白的相互作用。在模拟麦醇溶蛋白重复结构域的重组重复多肽（即（Pro-Gln-Gln-Pro-Tyr）8 上观察到类似的结合行为。综上所述，这些结果表明，PINA 与单体麦醇溶蛋白的相互作用形成了一个成核点，导致其他麦醇溶蛋白的聚集，这种现象可以防止贮藏醇溶蛋白与淀粉颗粒进一步相互作用。因此，应该根据先前的观察结果来解决多聚谷氨酰胺-醇溶蛋白相互作用对籽粒硬度的作用，这些观察结果突出了贮藏醇溶蛋白和多聚谷氨酰胺的类似亚细胞途径。
