Kimura Shizuka, Higashino Yuki, Kitao Yuki, Masuda Taro, Urade Reiko
BMC Plant Biol. 2015 Mar 4;15:73. doi: 10.1186/s12870-015-0460-2.
The major wheat seed proteins are storage proteins that are synthesized in the rough endoplasmic reticulum (ER) of starchy endosperm cells. Many of these proteins have intra- and intermolecular disulfide bonds. In eukaryotes, the formation of most intramolecular disulfide bonds in the ER is thought to be catalyzed by protein disulfide isomerase (PDI) family proteins. The cDNAs that encode eight groups of bread wheat (Triticum aestivum L.) PDI family proteins have been cloned, and their expression levels in developing wheat grains have been determined. The purpose of the present study was to characterize the enzymatic properties of the wheat PDI family proteins and clarify their expression patterns in wheat caryopses.
PDI family cDNAs, which are categorized into group I (TaPDIL1Aα, TaPDIL1Aβ, TaPDIL1Aγ, TaPDIL1Aδ, and TaPDIL1B), group II (TaPDIL2), group III (TaPDIL3A), group IV (TaPDIL4D), and group V (TaPDIL5A), were cloned. The expression levels of recombinant TaPDIL1Aα, TaPDIL1B, TaPDIL2, TaPDIL3A, TaPDIL4D, and TaPDIL5A in Escherichia coli were established from the cloned cDNAs. All recombinant proteins were expressed in soluble forms and purified. Aside from TaPDIL3A, the recombinant proteins exhibited oxidative refolding activity on reduced and denatured ribonuclease A. Five groups of PDI family proteins were distributed throughout wheat caryopses, and expression levels of these proteins were higher during grain filling than in the late stage of maturing. Localization of these proteins in the ER was confirmed by fluorescent immunostaining of the immature caryopses. In mature grains, the five groups of PDI family proteins remained in the aleurone cells and the protein matrix of the starchy endosperm.
High expression of PDI family proteins during grain filling in the starchy endosperm suggest that these proteins play an important role in forming intramolecular disulfide bonds in seed storage proteins. In addition, these PDI family proteins that remain in the aleurone layers of mature grains likely assist in folding newly synthesized hydrolytic enzymes during germination.
小麦种子的主要蛋白质是贮藏蛋白，它们在淀粉胚乳细胞的糙面内质网（ER）中合成。这些蛋白质中有许多具有分子内和分子间二硫键。在真核生物中，内质网中大多数分子内二硫键的形成被认为是由蛋白质二硫键异构酶（PDI）家族蛋白催化的。已经克隆了编码八组面包小麦（Triticum aestivum L.）PDI家族蛋白的cDNA，并测定了它们在发育中的小麦籽粒中的表达水平。本研究的目的是表征小麦PDI家族蛋白的酶学特性，并阐明它们在小麦颖果中的表达模式。
克隆了分为第I组（TaPDIL1Aα、TaPDIL1Aβ、TaPDIL1Aγ、TaPDIL1Aδ和TaPDIL1B）、第II组（TaPDIL2）、第III组（TaPDIL3A）、第IV组（TaPDIL4D）和第V组（TaPDIL5A）的PDI家族cDNA。从克隆的cDNA中确定了重组TaPDIL1Aα、TaPDIL1B、TaPDIL2、TaPDIL3A、TaPDIL4D和TaPDIL5A在大肠杆菌中的表达水平。所有重组蛋白均以可溶形式表达并纯化。除TaPDIL3A外，重组蛋白对还原和变性的核糖核酸酶A表现出氧化重折叠活性。五组PDI家族蛋白分布在整个小麦颖果中，这些蛋白在灌浆期的表达水平高于成熟后期。通过对未成熟颖果的荧光免疫染色证实了这些蛋白在内质网中的定位。在成熟籽粒中，五组PDI家族蛋白保留在糊粉层细胞和淀粉胚乳的蛋白质基质中。
淀粉胚乳灌浆期PDI家族蛋白高表达表明这些蛋白在种子贮藏蛋白分子内二硫键形成中起重要作用。此外，保留在成熟籽粒糊粉层中的这些PDI家族蛋白可能在萌发过程中协助新合成的水解酶折叠。
