Deposition Of Protein

From a nutritional point of view, proteins make up the second major component (about 10% of the dry weight) of the barley grain, but contain little of the essential amino acids lysine, methionine, tryptophan and threonine. Although barley is the poor cousin of wheat when considering the baking value of its storage proteins, these proteins are nonetheless major contributors to the functional properties of the grain and influence its malting performance. The deposition of starch and storage proteins is in addition tightly linked during grain filling, so that mutations affecting either starch or protein biosynthesis have a pleiotropic effect on accumulation of the other component in the endosperm. For instance, the "high lysine" mutant Riso 1508, which affects a regulatory locus to abolish trypsin inhibitor CMe expression (94), develops fewer aleurone cells (17), prematurely ceases enlargement of the starchy endosperm cells (18), and also deposits larger but fewer B-granules of starch (93). Hence, we shall consider briefly what is known of protein accumulation in barley.

5.1. Source of nitrogen for grain filling

The developing endosperm is supplied with both carbohydrates and nitrogen by the maternal tissues. The nitrogen supplied by the maternal tissue is mobilized not only from nutrients taken up from the soil, but also from proteins that are hydrolyzed in senescing leaves and in the degrading parts of the ovule (95). Changes in the proteinase complement during leaf senescence may be related to the regulation of nitrogen mobilization in barley (96); cysteine proteinases have been shown to be involved in leaf senescence in both monocotyledonous and dicotyledonous plants (97, 98). Two proteinases, nucellin (99) and nucellain (100), have been identified to be also present during autolysis of the nucellus in barley. Nucellin is an aspartic proteinase, whereas nucellain, localized in the cell wall, shows homology to an vacuolar-processing enzyme of castor bean (Ricinus communis). The exact hydrolytic roles of these proteases, in leaves or nucella, have however not yet been determined. Therefore the regulatory mechanisms involved in the breaking down of abundant proteins, such as ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco), remain to be elucidated. During seed maturation, leaf senescence proceeds in a sequential manner from the lowermost leaves to the higher leaves, but detailed knowledge of the regulatory genes and steps is needed before an understanding of this process can be reached.

5.2. Protein content of the endosperm

Barley storage proteins are mainly prolamins, a polymorphic mixture of proteins with Mr values between 30 000 and 90 000 that are deposited into the starchy endosperm. The ones found in barley can, based on their amino acid sequences, be classified into three groups, the S-rich, the Spoor, and the high molecular weight (HMW) prolamins. The major (8090%) fraction of the prolamins belong to the S-rich class, which includes both polymeric and monomeric components and which constitutes two families of storage proteins, the B and the y-hordeins. The C hordeins are S-poor prolamins, whereas the D hordeins are HMW prolamins (101). In developing grains, the hordeins are transported through the endoplasmic reticulum (ER) to the vacuole. Their synthesis is associated with the up-regulation of proteins involved in the maturation of secretory proteins within the ER-lumen (HSP70 and protein disulfide isomerase) as well as with the up-regulation of proteins that are involved in the transport of secretory proteins (Secl8p and Sarlp) from the ER to the cis-Golgi (102). In addition to the hordeins, the protein bodies of the starchy endosperm contain granular inclusions that do not react with antibodies to hordeins.

These inclusions could correspond to the 12S 6-globulins that have been localized to the protein bodies of the starchy endosperm in wheat (103).

Unlike the cells of the starchy endosperm, the cells of the aleurone layer do not contain starch or hordeins. Instead, the aleurone cells contain as the major storage protein a 7S globulin (104), which become deposited into discrete protein storage vacuoles (101, 103). The transport mechanisms involved in targeting prolamins and globulins to the vacuoles may differ, but in barley there is no evidence for a pathway from the ER to the vacuole that would not involve the Golgi (105, 106).

In addition to the major storage proteins, other proteins are present. These seem to play a protective role against insects, fungi, and bacteria in the resting seeds. Included in this second category of proteins localized in the endosperm are the chymotryptic inhibitors CI-1 and CI-2 that inhibit chymotrypsin, the trypsin/ a-amylase inhibitors that inhibit serine proteases and heterologous a-amylases, the hordothionins that interfere with redox systems, the endochitinases C and T that hydrolyze chitin, and also inhibitors of protein synthesis (103, 107, 108).

Although the bulk of the hydrolytic enzymes needed for efficient mobilization of both the carbohydrate and the protein reserves of the grain is synthesized only upon germination, some hydrolytic enzymes accumulate already during seed maturation. Included in this group are carboxypeptidase II (109) and p-amylase, both of which accumulate in the starchy endosperm during seed maturation (110), and an aspartic proteinase (111) which is deposited into protein storage vacuoles of the resting scutelium and aleurone layer (112, 113). In addition, cysteine proteinases have been localized, in small amounts, to protein storage vacuoles before gibberellin treatment (113). The relative amount of these proteins is low in comparison to the hordeins, Nevertheless, a single enzyme, p-amylase, alone constitutes 1-2% of the total grain proteins (110). The activity of these enzymes is thought to be down-regulated until seed germination by mechanisms such as covalent linkage to an inactivating protein (in the case of p-amylase, the linkage is to protein Z), intracellular localization, or changes in pH (114, 115).

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