The expression of hydrolytic enzymes in germinating barley grain

Abstract

Modification of the barley grain endosperm in germination is fundamental to successful plant growth but also has important ramifications for down-stream industrial uses of barley, particularly in the malting and brewing industries. There are a battery of enzymes that are involved in the modification process but the sites of synthesis and action of only of a few of these have been described in detail and most have only been studied in isolated tissues. The development of a sensitive and robust in situ mRNA hybridization (ISH) procedure, which allows the detection of specific transcripts representing proteins in grain sections is described here. This first required the optimization of grain fixation, embedding and sectioning procedures as well as the adaptation of a staining method using calcofluor white to accurately assess the modification state of each grain prior to processing. Once these technical parameters were established, the non-radioactive ISH protocol was developed to allow the detection of transcripts of (1,3;1,4)-β-glucanases, (1→4)-β-endo-xylanases, limit dextrinase and limit dextrinase inhibitor in grain sections of the two barley cultivars Sloop and Himalaya. The panel of enzymes selected for study covers several aspects of grain modification, such as (1,3;1,4)-β-glucan, xylan and starch breakdown, which are all important for successful grain germination. The successful ISH technique proved sensitive enough to discriminate between the (1,3;1,4)-β-glucanase and xylanase isoenzymes and clearly defined whether the transcipts for these enzymes were synthesized in both a tissue-specific manner and a fixed temporal sequence during grain germination. The use of monoclonal antibodies specific for the two (1,3;1,4)-β-glucanase isoforms in a related immunolabelling procedure, using the same fixed grains, also allowed the patterns of transcript and protein appearance to be correlated. As expected, use of the ISH method showed that the transcripts of the (1,3;1,4)-β-glucanase, xylanase and limit dextrinase inhibitor genes are variously found in the aleurone cells, the starchy endosperm tissue and the scutellum. However, there were also substantial amounts of transcript detected in the tissues of the growing embryo which suggests that these enzymes may also contribute substantially to early seedling development.