The importance of oxygen availability in two plant-based bioprocesses: hairy root cultivation and malting: Dissertation

The main aim of this work was to examine the role of oxygen availability in two model processes: the cultivation of Agrobacterium rhizogenes-induced hairy roots of the medicinal plant Egyptian henbane (Hyoscyamus muticus), and barley (Hordeum vulgare) malting. This was accomplished by transferring the vhb gene encoding bacterial haemoglobin (VHb) from Vitreoscilla to H. muticus and to barley with the specific goals of improving the growth and alkaloid production of H. muticus hairy roots and the germination rate of barley. In H. muticus hairy roots, heterologous vhb expression and an increase in aeration both improved the growth, but not the specific hyoscyamine production of the hairy roots. This suggests that oxygen availability may not be a limiting factor in hyoscyamine synthesis. The effect of VHb on the alkaloid profile of H. muticus hairy roots was not identical to that of aeration, indicating that the physiological effects of VHb are probably not related directly to its ability to increase the intracellular effective oxygen concentration. Although VHb production did not have a statistically significant effect on hyoscyamine production due to wide somaclonal variation, the highest production levels were found among the VHb-lines. A problem related to vhb expression was the tendency of hairy roots to dedifferentiate to callus. This effect probably counteracted the positive effects of vhb expression, because root dedifferentiation leads to impaired alkaloid production. Barley plants expressing the vhb gene did not react to oxygen deficiency by increasing the alcohol dehydrogenase (ADH) activity in the roots, unlike the control plants. This could be due to the ability of VHb to supplement the role of ADH in the recycling of NADH and maintaining glycolysis. In contrast to previous findings, we found that constitutive vhb expression did not improve the germination rate of barley kernels. On the contrary, VHb even retarded germination slightly. Moreover, VHb restricted root growth in young barley seedlings. These effects are probably related to the ?NO dioxygenase activity of VHb. Because nitric oxide (NO) has both cytotoxic and stimulating properties, the effect of vhb expression in plants may depend on the level and role of endogenous NO in the conditions studied. In conditions involving excess NO production, such as severe oxygen deficiency, VHb can protect plant cells from the adverse effects of NO. However, in conditions in which NO plays an important role as a signal molecule, such as germination and root growth, vhb expression may even have slightly adverse effects on growth and development. Another aim of the study was to verify the hypothesis that barley embryos suffer from oxygen deficiency in the malting process, regardless of aeration. This was accomplished by studying the effect of aeration on the production of the inducible isoenzymes ADH2 and ADH3, ADH activity, ethanol production and -amylase activity. Furthermore, the effect of the indigenous microbial community of the barley kernel on embryo oxygen deficiency was studied by modifying the microbial populations in the malting process. Neither aeration nor a reduction in grain microbes alleviated the oxygen deficiency of the barley embryo. An improvement in germination was observed after restriction of bacteria, but this was probably a consequence of facilitated root emergence. Many of the bacterial species dominating in the malting process can produce exopolymeric substances that may form a physical barrier restricting root emergence. A certain degree of oxygen deficiency in the embryo appears to be an inevitable stage in barley germination. The recent results of other research groups showing that NO is produced in plant cells under oxygen deficiency and that NO stimulates seed germination, together with the present results showing that vhb expression slows down barley germination slightly, imply that embryo oxygen deficiency is in fact beneficial or even necessary for germination. Although aeration cannot and need not overcome the oxygen deficiency of the barley embryo, the need for aeration increases as the malting process proceeds. Aeration not only introduces the oxygen needed for the completion of germination, enzyme synthesis and other post-germinative events, but also removes CO2 that would otherwise inhibit modification of the grain.