Subtilase activity and gene expression during germination and seedling growth in barley.

Proteases play a main role in the mobilization of storage proteins during seed germination. Until today, there is little information about the involvement of serine proteases, particularly subtilases, in the germination of barley grains. The aims of the present work were to study the contribution of serine proteases to the total proteolytic activity induced during germination of barley grains and evaluate the specific involvement of subtilases in this process. Proteolytic activity assayed against azocasein in the presence of specific inhibitors, showed that serine proteases contributed between 10 and 20% of total activity along germination. Subtilase activity increased from day 1 after imbibition with a peak between days 4-5. Moreover, in vivo determination of subtilase activity in germinating grains revealed increasing activity along germination mainly localized in the seed endosperm and developing rootlets. Finally, the expression of 19 barley genes encoding subtilases was measured by real time PCR during germination. Three of the analyzed genes increased their expression along germination, five showed a transient induction, one was down-regulated, nine remained unchanged and one was not expressed. The present work demonstrates the involvement of subtilases in germination of barley grains and describes the positive association of eight subtilase genes to this process.

A novel Burkholderia ambifaria strain able to degrade the mycotoxin fusaric acid and to inhibit Fusarium spp. growth.

Fusaric acid (FA) is a fungal metabolite produced by several Fusarium species responsible for wilts and root rot diseases of a great variety of plants. Bacillus spp. and Pseudomonas spp. have been considered as promising biocontrol agents against phytopathogenic Fusarium spp., however it has been demonstrated that FA negatively affects growth and production of some antibiotics in these bacteria. Thus, the capability to degrade FA would be a desirable characteristic in bacterial biocontrol agents of Fusarium wilt. Taking this into account, bacteria isolated from the rhizosphere of barley were screened for their ability to use FA as sole carbon and energy source. One strain that fulfilled this requirement was identified according to sequence analysis of 16S rRNA, gyrB and recA genes as Burkholderia ambifaria. This strain, designated T16, was able to grow with FA as sole carbon, nitrogen and energy source and also showed the ability to detoxify FA in barley seedlings. This bacterium also exhibited higher growth rate, higher cell densities, longer survival, higher levels of indole-3-acetic acid (IAA) production, enhanced biofilm formation and increased resistance to different antibiotics when cultivated in Luria Bertani medium at pH 5.3 compared to pH 7.3. Furthermore, B. ambifaria T16 showed distinctive plant growth-promoting features, such as siderophore production, phosphate-solubilization, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, in vitro antagonism against Fusarium spp. and improvement of grain yield when inoculated to barley plants grown under greenhouse conditions. This strain might serve as a new source of metabolites or genes for the development of novel FA-detoxification systems.