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In the present manuscript, we report on the proteolytic enzymes acting in the Araucaria bidwillii megagametophyte throughout seed germination. At seed maturity the megagametophyte contains a bulk of reserves for the growing embryo, thus representing the major storage tissue of the bunya pine seed. Soon after seed germination the megagametophyte undergoes storage protein mobilization, degenerating as a no longer needed tissue by the late germinative stages. By using in-solution and in-gel assays, and mass spectrometric analyses we detected exopeptidases and proteinases differently active in this tissue at selected germinative stages, and obtained preliminary data on the nature of an endopeptidase active at the late stages. Early germination stages were characterized by aminopeptidase and aspartic, metallo and cysteine proteinase activities; carboxypeptidases and serine proteinases became highly active by the late stages. Partial sequencing of a protein responsible for late stage serine peptidase activity sensitive to the caspase-6 inhibitor, showed a set of amino acid sequences with various degrees of identity with various plant subtilisin-like serine proteinases. The participation of the early stage proteases in the storage protein mobilization and the involvement of the late stage proteases in the megagametophyte cell death are proposed and discussed.

The megagametophyte of the Araucaria bidwillii seed is a storage tissue that surrounds and feeds the embryo. When all its reserves are mobilized, the megagametophyte degenerates as a no longer needed tissue. In this work we present a biochemical and a cytological characterization of the megagametophyte cell death. The TUNEL assay showed progressive DNA fragmentation throughout the post-germinative stages, while DNA electrophoretic analysis highlighted a smear as the predominant pattern of DNA degradation and internucleosomal DNA cleavage only for a minority of cells at late post-germinative stages. Cytological investigations at these stages detected profound changes in the size and morphology of the megagametophyte nuclei. By using in vitro assays, we were able to show a substantial increase in proteolytic activities, including caspase-like protease activities during the megagametophyte degeneration. Among the caspase-like enzymes, caspase 6- and 1-like proteases appeared to be the most active in the megagametophyte with a preference for acidic pH. On the basis of our results, we propose that the major pathway of cell death in the Araucaria bidwillii megagametophyte is necrosis; however, we do not exclude that some cells undergo developmental programmed cell death.

The proteins belonging to the cereal trypsin/alpha-amylase inhibitor family are abundant water/salt-soluble flour proteins active against alpha-amylases from several seed parasites and pests and inactive against endogenous alpha-amylases. Three alpha-amylase inhibitor families have been described in cereals that vary in size and are differently expressed among Triticeae seeds. The present work investigates the presence of human salivary alpha-amylase inhibitors in emmer (Triticum dicoccon Schrank) flour. The isolation was obtained by a series of chromatography steps, and the purification progress was monitored through the inhibition of human salivary alpha-amylase activity. The purified fraction was subjected to protein sequencing by tandem mass spectrometry (MSMS) of the tryptic digests obtained after the sample separation on 2-DE. MSMS data indicated that the emmer alpha-amylase inhibitory fraction was composed of two newly identified proteins [emmer dimeric inhibitor 1 (EDI-1) and emmer dimeric inhibitor 2 (EDI-2)] sharing very high identity levels with related proteins from Triticum aestivum.

Seeds of bread wheat were incubated at 40 degrees C and 100% relative humidity for 0, 3, 4, 6, and 10 days. The effects of accelerated aging on seed germinability and some biochemical properties of flour (carotenoid, free radical, and protein contents and proteolytic activity) and gluten (free radical content and flexibility) were investigated. Seed germinability decreased during aging, resulting in seed death after 10 days. A progressive decrease of carotenoid content, in particular, lutein, was observed, prolonging the incubation, whereas the free radical content increased in both flour and gluten. A degradation of soluble and storage proteins was found, associated with a marked increase of proteolytic activity and a loss of viscoelastic properties of gluten. On the contrary, puroindolines were quite resistant to the treatment. The results are discussed in comparison with those previously obtained during accelerated aging of durum wheat seeds.

Accelerated aging was performed by incubation of wheat seeds at 40 degrees C and 100% relative humidity for 3, 4, 6, 10, and 14 days. The effects of the treatment on seed germinability and on several biochemical characteristics of flour (carotenoids, free radical and protein contents, and proteolytic activity) and gluten (free radical content and flexibility) were evaluated. A decrease of germinability was found during aging, the germination being completely inhibited after 14 days. The lutein content decreased gradually, without going to zero, while that of free radicals increased. A reduction of soluble proteins and a degradation of glutenins and gliadins were observed, associated with a substantial increase of protease activity and a decrease in gluten flexibility. The results were discussed in reference to those previously obtained by natural aging of wheat seeds of the same species and cultivar.

Asparaginyl-endopeptidase activity was detected in endosperms of maturing and germinating wheat seeds. The highest activity was found during maturation before the maximal accumulation of storage proteins. The enzyme activity then decreased in the dry seeds and increased again during germination. The increase of activity during germination required the presence of the embryo. In fact, the activity found in detached endosperms was lower than that found in attached ones. The localization at tissue level of the enzyme reveals differences between maturation and germination: the enzyme was about equally located in the aleurone layer and starchy endosperm during maturation, but solely in the aleurone layer during germination. The asparaginyl enzymes from maturing and germinating seeds had many similar properties, such as pH optimum, pH stability, thermal stability and sensitivity to thiol reagents and to thiol compounds. The results suggest that asparaginyl endopeptidases may be involved in the modification of proproteins of storage proteins during seed maturation and in the degradation of storage proteins deposited in the aleurone layer during germination. [ABSTRACT FROM AUTHOR]