Revisiting Rubisco as a Protein Substrate for Insect Midgut Proteases

Abstract

Gene fragments encoding the large subunit (LS) of Rubisco (RBCL) were cloned from various species of host plants of phytophagous Lepidoptera and expressed as recombinant proteins in Escherichia coli. Recombinant RBCLs were compared among each other along with casein and native Rubisco as proteinaceous substrates for measuring total midgut protease activities of fourth instar larvae of Helicoverpa armigera feeding on casein, Pieris brassicae feeding on cauliflower, and Antheraea assamensis feeding on Litsea monopetala and Persea bombycina. Cognate rRBCL (from the pertinent host plant species) substrates performed similar to noncognate rRBCL reflecting the conserved nature of encoding genes and the versatile use of these recombinant proteins. Casein and recombinant RBCL generally outperformed native Rubisco as substrates, except where inclusion of a reducing agent in the enzyme assay likely unfolded the plant proteins. Levels of total midgut protease activities detected in A. assamensis larvae feeding on two primary host species were similar, suggesting that the suite(s) of digestive enzymes in these insects could hydrolyze a plant protein efficiently. Protease activities detected in the presence of protease inhibitors and the reducing agent dithiothreitol (DTT) suggested that recombinant RBCL was a suitable protein substrate for studying insect proteases using in vitro enzyme assays and substrate zymography.

Midgut serine proteases and alternative host plant utilization in Pieris brassicae L.

“…Multiple trypsin substrates were employed as fourth instar P. brassicae larvae fed on cauliflower have multiple pH optima for total midgut protease activities detected using protein substrates viz. casein and rRBCL (Bhardwaj et al, 2014 ). Chymotrypsin activities were detected in the midgut samples using the fluorogenic substrate SAAPFAMC at pH 10 (Figure 2D ).…”

“…Total midgut protease activity was measured in field-collected fourth instar larvae feeding on mature leaves (ML) of cauliflower, cabbage, mustard, radish and T. majus . Rubiscolytic activities were detected using purified Rubisco from N. tabacum foliage after Bhardwaj et al ( 2014 ). Briefly, 1 μg protein was incubated with 200 μg of purified Rubisco in 100 mM Glycine-NaOH buffer, pH 10.…”

“…Enzyme only controls (without substrate), and enzyme and inhibitor controls (without substrate) were also included. Gelatinolytic zymograms were run according to the modified method of Lantz and Ciborowski ( 1994 ); Michaud ( 1998 ); Oppert et al ( 1998 ) and Bhardwaj et al ( 2014 ). Equal amounts of midgut extract from 4th instar larvae were applied to each lane of the activity gels.…”

“…Like other Lepidoptera, digestive serine proteases have been reported from various pierid larvae (Broadway, 1989a ; Broadway and Colvin, 1992 ; Liao et al, 2007 ; Zhou et al, 2008 ; Zibaee, 2012 ; Bhardwaj et al, 2014 ). Midgut trypsins and chymotrypsins can digest ingested plant tissues containing proteins (like Rubisco) that are sources of amino acids and energy essential for insect growth and development (Christeller et al, 1992 ; Woods and Kingsolver, 1999 ).…”

Ammonium and nitrate levels of soil inoculated with Azospirillum brasilense in maize

“…The major challenge currently in agricultural production is reducing the production cost, by keeping the yield level with minimized environmental impact (Bhardwaj et al, 2014). In this context, utilization of plant growth promoting rhizobacteria can be a great strategy for this purpose.…”