Overexpression of the protease inhibitor BoCPI-1 in broccoli delays chlorophyll loss after harvest and causes down-regulation of cysteine protease gene expression

• Protease activity is involved in the postharvest senescence of detached tissues.
• A cDNA clone for a broccoli cysteine protease inhibitor, BoCPI-1, was isolated.
• BoCPI-1 showed harvest-induced expression in detached broccoli heads.
• Overexpression of BoCPI-1 in transgenic broccoli reduced postharvest senescence.
• Overexpression of BoCPI-1 reduced the mRNA abundance of several cysteine proteases.

Papain-like cysteine proteases are involved in many physiological processes in vascular plants, including senescence and programmed cell death. Here we report the isolation of a cysteine protease inhibitor (BoCPI-1) from broccoli (Brassica oleracea var. italica), and characterise its role in regulating protease activity. Biochemical analysis showed BoCPI-1 had inhibitory activity against papain. Broccoli was genetically modified to overexpress BoCPI-1, and both azocasein assays (which provide a relatively crude measure of total protease activity), together with DCG-04 assays (which allow more targeted analysis of cysteine protease activity), were used to examine the function of the inhibitor during postharvest senescence. In broccoli heads, overexpression of BoCPI-1 reduced total protease activity, retained cellular soluble protein content and delayed the onset of postharvest senescence as measured by chlorophyll loss. Up-regulating the expression of BoCPI-1 resulted in a lower mRNA accumulation of five different senescence-associated cysteine protease genes (BoCP1, BoCP2, BoCP3, BoCP4, BoCP5). The link between the transcription of a cysteine protease inhibitor (a phytocystatin) and the transcription of the structurally unrelated cysteine proteases suggests that the changed cysteine protease mRNA accumulation patterns are the result of a feedback loop that is regulated by cellular protease activity.