Senescence is the final developmental stage of every plant organ, which leads to cell death. It is a highly regulated process, involving differential gene expression and outstanding increment in the rate of protein degradation. Senescence-associated proteolysis enables the remobilization of nutrients, such as nitrogen (N), from senescent tissues to developing organs or seeds. In addition to the nutrient recycling function, senescence-associated proteases are also involved in the regulation of the senescence process. Nearly, all protease families have been associated with some aspects of plant senescence, and numerous reports addressing the new identification of senescence-associated proteases are published every year. Here, we provide an updated report with the most recent information published in the field, focusing on senescence-associated proteases presumably involved in N remobilization.

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A senescence-specific protease accounting for almost 70% of the total peptide hydrolytic activity of protein extracts, was isolated from detached wheat leaves induced to senescence by incubation in the dark for 72 h. Purification to apparent homogeneity was performed by ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The enzymatic activity was followed by its ability to hydrolyse the synthetic peptide Suc-AAPF-pNA. SDS/PAGE and gel filtration analysis indicated that the enzyme was a dimer composed of two identical subunits of 59 kDa. The apparent K m and V max for the peptide were 1.18 mM and 2.27 mmol pNA mg À1 h À1 , respectively. The enzyme was active at pH values above 8.0 and remained active after heat treatment at 60 C for 10 min. It was inhibited by chymostatin, indicating that the enzyme possesses a chymotrypsin-like activity. Rubisco was readily hydrolysed by the purified protease. A sequenced internal fragment of 17 amino acids showed a high level of similarity (65-75% identity) with a highly conserved region of several plant subtilisin-like serine proteases. The absence of this enzymatic activity in fractionated extracts from non-senescent tissues suggests that it might play a role in the senescing process.

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The induction of two subtilisin-like proteases (P1 and P2) associated with stress-induced senescence in young plants was investigated in adult wheat plants during the grain-filling period. Western blot analysis of flag leaf extracts showed that P1 was induced very late in the life cycle of the plants (9 days post-anthesis) and that 7 days later it reached a 2.5-fold increase with respect to the initial value at anthesis. On the other hand, the P2 signal was already detected previous to anthesis and increased soon after anthesis, reaching a fourfold increase by the end of the grain-filling period. The induction of P1 and P2 temporally correlates with the degradation of the Rubisco small and large subunits in the flag leaf, as well as with nitrogen (N) accumulation in the ears. At the same time, a decrease in the endogenous concentration of the cytokinins isopentenyladenine and isopentenyladenosine (iP ? iPA) in the leaves was observed. In detached leaves senescing in the dark, the levels of both proteases were affected by 6-benzylaminopurine application: the induction of P1 was completely prevented, whereas the induction of P2 was reduced. Our findings demonstrate that both P1 and P2 are expressed in leaves of adult plants and are induced during natural senescence. These results enable us to postulate their participation in N remobilization to developing grains during monocarpic senescence and their regulation by a cytokinin-mediated mechanism.

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The widely distributed members of the Deg/HtrA protease family play an important role in the proteolysis of misfolded and damaged proteins. Here we show that the Deg protease rHhoA is able to degrade PsbO, the extrinsic protein of the Photosystem II (PSII) oxygen-evolving complex in Synechocystis sp. PCC 6803 and in spinach. PsbO is known to be stable in its oxidized form, but after reduction by thioredoxin it became a substrate for recombinant HhoA (rHhoA). rHhoA cleaved reduced eukaryotic (specifically, spinach) PsbO at defined sites and created distinct PsbO fragments that were not further degraded. As for the corresponding prokaryotic substrate (reduced PsbO of Synechocystis sp. PCC 6803), no PsbO fragments were observed. Assembly to PSII protected PsbO from degradation. For Synechocystis sp. PCC 6803, our results show that HhoA, HhoB, and HtrA are localized in the periplasma and/or at the thylakoid membrane. In agreement with the idea that PsbO could be a physiological substrate for Deg proteases, part of the cellular fraction of the three Deg proteases of Synechocystis sp. PCC 6803 (HhoA, HhoB, and HtrA) was detected in the PSII-enriched membrane fraction.

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.