Cysteine protease and cystatin expression and activity during soybean nodule development and senescence.

BACKGROUND: Nodules play an important role in fixing atmospheric nitrogen for soybean growth. Premature senescence of nodules can negatively impact on nitrogen availability for plant growth and, as such, we need a better understanding of nodule development and senescence. Cysteine proteases are known to play a role in nodule senescence, but knowledge is still fragmented regarding the function their inhibitors (cystatins) during the development and senescence of soybean nodules. This study provides the first data with regard to cystatin expression during nodule development combined with biochemical characterization of their inhibition strength. RESULTS: Seventy nine non-redundant cysteine protease gene sequences with homology to papain, belonging to different subfamilies, and several legumain-like cysteine proteases (vacuole processing enzymes) were identified from the soybean genome assembly with eighteen of these cysteine proteases actively transcribed during nodule development and senescence. In addition, nineteen non-redundant cystatins similar to oryzacystatin-I and belonging to cystatin subgroups A and C were identified from the soybean genome assembly with seven actively transcribed in nodules. Most cystatins had preferential affinity to cathepsin L-like cysteine proteases. Transcription of cystatins Glyma05g28250, Glyma15g12211, Glyma15g36180 particularly increased during onset of senescence, possibly regulating proteolysis when nodules senesce and undergo programmed cell death. Both actively transcribed and non-actively transcribed nodule cystatins inhibited cathepsin-L- and B-like activities in different age nodules and they also inhibited papain and cathepsin-L activity when expressed and purified from bacterial cells. CONCLUSIONS: Overlap in activities and specificities of actively and non-actively transcribed cystatins raises the question if non-transcribed cystatins provide a reservoir for response to particular environments. This data might be applicable to the development of strategies to extend the active life span of nodules or prevent environmentally induced senescence.

Proteolysis of recombinant proteins in bioengineered plant cells.

Plants are increasingly used as alternative expression hosts for the production of recombinant proteins offering many advantages including higher biomass and the ability to perform post-translational modifications on complex proteins. Key challenges for optimized accumulation of recombinant proteins in a plant system still remain, including endogenous plant proteolytic activity, which may severely compromise recombinant protein stability. Several strategies have recently been applied to improve protein stability by limiting protease action such as recombinant protein production in various sub-cellular compartments or application of protease inhibitors to limit protease action. A short update on the current strategies applied is provided here, with particular focus on sub-cellular sites previously selected for recombinant protein production and the co-expression of protease inhibitors to limit protease activity.

Isolation and identification of a novel chlorophenol from a cell suspension culture of Helichrysum aureonitens.

A novel chlorophenol, 4-chloro-2-(hepta-1,3,5-triyn-1-yl)-phenol (1), was isolated as the major phenolic compound from the cells of Helichrysum aureonitens suspension cultures. Compound 1 has been proposed to be an intermediate in the acetylene biosynthetic pathway of other acetylenic compounds in Helichrysum spp. The ethanol extract of cell suspension cultures and compound 1 were evaluated for their cytotoxicity against monkey kidney Vero (Vero cells) and human prostate epithelial carcinoma (DU145) cell lines, also, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Mycobacterium tuberculosis H37Rv were determined as well.