1 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark2 Department of Systems Biology, Technical University of Denmark
Heterologous expression of barley limit dextrinase inhibitor
Cereal grains are vital components of our diet, and therefore understanding of the biology of breakage of dormancy and initiation of germination very important. These processes are far from fully understood, despite extensive studies, and no specific markers for germination have been identified at the protein or the DNA level. In addition, germination of barley seeds is of interest for the brewing industry since this process corresponds to the steeping process that starts the industrial malting. In the present study a proteomics approach was employed to understand the initial changes in the water soluble protein composition of the barley seed upon imbibition and the following events that occur until to 72 h post imbibition (PI). 2D gel electrophoresis of proteins extracted from dissected barley seeds tissues during germination (0-24 h) and the subsequent radicle elongation (24-72 h) describes spatio-temporal variations in the protein patterns. Seeds from 8 time points (0, 4, 12, 24, 36, 52, 60, and 72 h PI) were dissected into embryo, aleurone layer and endosperm and small scale protein extractions enabled us to obtain good resolution 2D gels. The 2D gels were compared between the time points and the different tissues and protein spots of changing intensity were tracked and identified by in-gel digestion, mass spectrometry, and database searches. Forty-eight different proteins in 79 protein spots were tracked through-out the processes and the distribution of these protein spots differed among embryo, aleurone, and endosperm emphasizing individual tissue functionalities. The most significant changes occurred in the two living tissues, namely the embryo and the aleurone layer, both of which resume metabolic activity upon imbibition. The earliest and the largest number of changes were observed in the embryo. This is in contrast to the endosperm which serves as storage reserve, where the hydrolytic enzymes, produced in the aleurone layer, take action in order to provide nutrients for the embryo growth. β-Type proteasome subunit and a fragment of heat shock protein 70 were identified at 4 h PI as the first proteins to appear in embryo together with changes in the spot pattern of desiccation stress related proteins such as late embryonic abundant (LEA) proteins and abscisic acid induced proteins. After 12 h well-known desiccation stress related proteins and other proteins (putative embryonic protein and abscisic acid induced protein), that we suggest are involved in desiccation, rapidly disappear. Other proteins including several redox-related proteins, which are involved in protection against water stress and reactive oxygen species, changed in abundance at the end of germination and start of radicle elongation. These proteins had different tissue distribution and most of them either remained constant or decreased in abundance during radicle elongation, except for ascorbate peroxidase (APX) which was only present in the germinating barley embryo after 24 h. Other proteins involved in osmotic and salinity stress, storage breakdown, folding, and housekeeping were identified and tracked through 72 h PI. APX and the other enzymes involved in the ascorbate-glutathione cycle (dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione reductase) were thought to have an important function during germination and radicle elongation, since the high energy demand upon imbibition generates reactive oxygen species, especially in the metabolically active and dividing embryo tissue. These four enzymes were subjected to activity assays carried out using barley embryo protein extracts and the activity profiles were compared IV to the 2D gel patterns. This gave both concordant and deviating enzymes activity and 2D gel spot intensities during radicle elongation. APX activity detection in zymograms and gene expression analysis indicated that at least two APX isoforms are present in the germinated embryo extract. The APX mRNA of both isoforms were present in the dry mature seed, whereas increasing activity was only observed after 36 h, which supports that the ascorbate-glutathione cycle is not functioning in the desiccated seed, but that the mRNA is ready for translation. Both APX and DHAR are suggested targets of Trx h-linked redox regulation, and this is discussed based on the present results, although it was not possible to detect changes in the dehydroascorbate reductase activity by addition of reduced, recombinant Trx h to the embryo extract. Post-translational modification of APX was further studied by mass spectrometry based on the 2D gel pattern of APX in germinated barley embryos, which indicated the presence of several APX forms with different pI values. The barley limit dextrinase inhibitor (LDI) is thought to be at least partly responsible for the presence of non-fermentable branched dextrins (α-limit dextrins) in barley malt by inhibiting the starch debranching enzyme limit dextrinase (LD). By producing the low abundant LDI in a heterologous host organism, the aim was to characterize the interaction between LD and LDI and the influence of Trx h on the interaction. Trx is suggested to reduce LDI and thereby indirectly increasing the activity of LD during malting. LDI cDNA was cloned from developing barley seeds and several vector constructs were made for heterologous expression; however, LDI was only successfully produced as a fusion protein with E. coli Trx h attached to the N-terminus. The fusion protein showed 16 fold lower activity than LDI purified from barley seeds. By removing the Trx tag by the endoprotease, enteropeptidase, the inhibitory activity further decreased 3-10 fold. This activity decrease was probably caused by unspecific cleavage by enteropeptidase in the LDI sequence due to non-native folding.