Abstract
Barley (Hordeum vulgare) is one of the major food sources for humans and forage sources for animal livestock. The average grain protein content (GPC) of barley ranges between 8 and 12%. Barley hordeins (i.e., prolamins) account for more than 50% of GPC in mature seeds and are important for both grain and flour quality. Barley endosperm is structured into three distinct cell layers: the starchy endosperm, which acts essentially as storage tissue for starch; the subaleurone, which is characterized by a high accumulation of seed storage proteins (SSPs); and the aleurone, which has a prominent role during seed germination. Prolamins accumulate in distinct, ER-derived protein bodies (PBs) and their trafficking route is spatio-temporally regulated. The protein disulfide isomerase (PDI) has been shown to be involved in PB formation. Here, we unravel the spatio-temporal proteome regulation in barley aleurone, subaleurone, and starchy endosperm for the optimization of end-product quality in barley. We used laser microdissection (LMD) for subsequent nanoLC-MS/MS proteomic analyses in two experiments: in Experiment One, we investigated the proteomes of dissected barley endosperm layers at 12 and at ≥20 days after pollination (DAP). We found a set of 10 proteins that were present in all tissues at both time points. Among these proteins, the relative protein abundance of D-hordein, B3-hordein and HvPDIL1-1 significantly increased in starchy endosperm between 12 and ≥20 DAP, identifying the starchy endosperm as putative major storage tissue. In Experiment Two, we specifically compared the starchy endosperm proteome at 6, 12, and ≥20 DAP. Whereas the relative protein abundance of D-hordein and B3-hordein increased between 6 and ≥20 DAP, HvPDIL1-1 increased between 6 and 12 DAP, but remained constant at ≥20 DAP. Microscopic observations showed that these relative protein abundance alterations were accompanied by additional localization of hordeins at the periphery of starch granules and a partial re-localization of HvPDIL1-1 from PBs to the periphery of starch granules. Our data indicate a spatio-temporal regulation of hordeins and HvPDIL1-1. These results are discussed in relation to the putative role of HvPDIL1-1 in end-product quality in barley.
Highlights
Barley is a cereal crop mainly used for food, animal feed and beverage production
laser microdissection (LMD) enables the study of the proteome of dissected aleurone, subaleurone, and starchy endosperm at different developmental time-points making it very useful in the study of spatio-temporal regulated proteins in the heterogeneous tissue of barley endosperm
We provide data that identifies the starchy endosperm as the most prominent storage tissue for seed storage protein (SSP) accompanied by a high abundance of endoplasmic reticulum (ER) structures and the accumulation of HvPDIL1-1
Summary
Barley is a cereal crop mainly used for food, animal feed and beverage production. Barley grain texture and/or grain protein content (GPC) is an important quality determinant for end uses such as malt production, animal feed as well as food (Cai et al, 2013). Cereal seed proteins can be classified according to their solubility into four groups: albumins, globulins, prolamins, and glutelin. Cereal seed proteins are classified into two groups according to their biological function: seed storage proteins (SSPs) and non-storage proteins (Shewry and Tatham, 1990). Prolamins are termed hordeins, and account for more than 50% of the total protein amount in mature seeds (Shewry and Tatham, 1990; Shewry, 1999). B and C hordeins together account for over 95% of the total hordein content (Rahman et al, 1984)
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