Protease inhibitors are a promising complement to Bt toxins for the development of insect-resistant transgenic crops, but their limited specificity against proteolytic enzymes and the ubiquity of protease-dependent processes in living organisms raise questions about their eventual non-target effects in agroecosystems. After a brief overview of the main factors driving the impacts of insect-resistant transgenic crops on non-target organisms, the possible effects of protease inhibitors are discussed from a multitrophic perspective, taking into account not only the target herbivore proteases but also the proteases of other organisms found along the trophic chain, including the plant itself. Major progress has been achieved in recent years towards the design of highly potent broad-spectrum inhibitors and the field deployment of protease inhibitor-expressing transgenic plants resistant to major herbivore pests. A thorough assessment of the current literature suggests that, whereas the non-specific inhibitory effects of recombinant protease inhibitors in plant food webs could often be negligible and their ‘unintended’ pleiotropic effects in planta of potential agronomic value, the innocuity of these proteins might always remain an issue to be assessed empirically, on a case-by-case basis.

Summary: Recombinant proteins face major constraints along the plant cell secretory pathway, including proteolytic processing compromising their structural integrity. Here, we demonstrate the potential of protease inhibitors as in situ stabilizing agents for recombinant proteins migrating towards the leaf apoplast. Genomic data for Arabidopsis, rice and Nicotiana spp. were assessed to determine the relative incidence of protease families in the cell secretory pathway. Transient expression assays with the model platform Nicotiana benthamiana were then performed to test the efficiency of protease inhibitors in stabilizing proteins targeted to the apoplast. Current genomic data suggest the occurrence of proteases from several families along the secretory pathway, including A1 and A22 Asp proteases; C1A and C13 Cys proteases; and S1, S8 and S10 Ser proteases. In vitro protease assays confirmed the presence of various proteases in N. benthamiana leaves, notably pointing to the deposition of A1‐ and S1‐type activities preferentially in the apoplast. Accordingly, transient expression and secretion of the A1/S1 protease inhibitor, tomato cathepsin D inhibitor (SlCDI), negatively altered A1 and S1 protease activities in this cell compartment, while increasing the leaf apoplast protein content by ∼45% and improving the accumulation of a murine diagnostic antibody, C5‐1, co‐secreted in the apoplast. SlCYS9, an inhibitor of C1A and C13 Cys proteases, had no impact on the apoplast proteases and protein content, but stabilized C5‐1 in planta, presumably upstream in the secretory pathway. These data confirm, overall, the potential of protease inhibitors for the in situ protection of recombinant proteins along the plant cell secretory pathway.

The expression of clinically useful proteins in plants has been bolstered by the development of high-yielding systems for transient protein expression using agroinfiltration. There is a need now to know more about how host plant development and metabolism influence the quantity and quality of recombinant proteins. Endogenous proteolysis is a key determinant of the stability and yield of recombinant proteins in plants. Here we characterised cysteine (C1A) and aspartate (A1) protease profiles in leaves of the widely used expression host Nicotiana benthamiana, in relation with the production of a murine IgG, C5-1, targeted to the cell secretory pathway. Agroinfiltration significantly altered the distribution of C1A and A1 proteases along the leaf age gradient, with a correlation between leaf age and the level of proteolysis in whole-cell and apoplast protein extracts. The co-expression of tomato cystatin SlCYS8, an inhibitor of C1A proteases, alongside C5-1 increased antibody yield by nearly 40% after the usual 6-days incubation period, up to ∼3 mg per plant. No positive effect of SlCYS8 was observed in oldest leaves, in line with an increased level of C1A protease activity and a very low expression rate of the inhibitor. By contrast, C5-1 yield was greater by an additional 40% following 8- to 10-days incubations in younger leaves, where high SlCYS8 expression was maintained. These findings confirm that the co-expression of recombinant protease inhibitors is a promising strategy for increasing recombinant protein yields in plants, but that further opportunity exists to improve this approach by addressing the influence of leaf age and proteases of other classes.

Summary: We reported earlier the potential of tomato cathepsin D inhibitor (SlCDI) as an in‐built stabilizing agent for the protection of recombinant proteins in transgenic plant leaf crude extracts (Plant Biotechnol J.4, 359–368). Here we document the potential of SlCDI for the in situ protection of proteins in potato leaves. Total protein assays with control and SlCDI‐expressing potato lines indicated a positive impact of slcdi transgene expression on leaf protein content, with a mean relative increase of 35%–40% depending on the light regime. Out of ∼700 proteins detected on 2‐D gels, only 20 exhibited a significantly altered level on a protein‐specific basis, whereas most proteins were up‐regulated on a leaf fresh weight basis, albeit at variable rates. Quantitative reverse trancriptase‐PCR assays for rubisco activase showed similar transcript levels in leaves of test and control lines despite protein levels increased by two‐ to threefold in SlCDI‐expressing lines. These observations, along with the unrelated biological functions assigned to MS‐identified proteins up‐regulated in leaves and protease assays showing slightly increased proteasome activity in protein extracts of SlCDI‐expressing lines, suggest a general, proteasome‐independent protein stabilizing effect of SlCDI in planta. Transient expression assays with human α₁‐antichymotrypsin also showed a stabilizing effect for SlCDI on heterologous proteins, leading to net levels of the human protein increased by ∼2.5‐fold in SlCDI‐expressing plants. These data illustrate, overall, the potential of SlCDI as an in vivo protein‐stabilizing agent in transgenic plant systems, useful to improve protein levels and recombinant protein accumulation.