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

Highlights

• 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.

Abstract

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.

Introduction

The postharvest life of broccoli is determined largely by the rate of loss of dark green colour, and is observed as yellowing due to chlorophyll loss from the florets (Costa et al., 2005). The act of harvest separates the head tissue from the parent plant and initiates premature senescence, a genetically programmed process which in broccoli involves a range of genes involved in multiple metabolic pathways (Chen et al., 2008). It is generally accepted that senescence progresses through three phases: (i) initiation, induced by environmental, hormonal or other endogenous signals, (ii) a degenerative phase, resulting in large-scale hydrolysis and mobilisation of macromolecules, particularly proteins or chlorophylls leading to yellowing, and (iii) the terminal phase, with the irreversible loss of cell integrity and death of plant tissues (Buchanan-Wollaston and Ainsworth, 1997, Yoshida, 2003, Desclos et al., 2009).

Proteolysis is indispensable for the maintenance and survival of an organism: an estimated 2% of the genes in higher organisms encode proteolytic enzymes that are involved in a range of plant developmental processes (Barrett and Rawlings, 2001). Due to the potentially damaging effects of proteases, their activities are strictly controlled (Rawlings et al., 2004). This may be achieved through differences in transcription, post-translational processing, subcellular compartmentalisation, substrate specificity, and the presence and activity of inhibitor proteins. In the latter phases of senescence, proteolysis provides a large pool of cellular nitrogen for recycling via ubiquitin-dependent, chloroplast and vacuolar degradative pathways (Vierstra, 1996). However, specific tightly regulated proteolytic activity may also have a role in regulating the onset and rate of senescence in the initial phases, similar to that proposed by Solomon et al. (1999) for the regulation of programmed cell death (PCD) following oxidative stress and pathogen attack.

Plant genomes are thought to encode approximately 140 cysteine proteases that belong to 15 families and at least 5 clans (Barrett and Rawlings, 2001). The activity of cysteine proteases has been associated with senescence in a range of plant species and tissues (Roberts et al., 2012), including broccoli where at least five different senescence-associated cysteine proteases have been characterised (Page et al., 2001, Coupe et al., 2003, Wang et al., 2004, Eason et al., 2005). Cysteine protease inhibitors (phytocystatins) act to co-regulate proteolysis and cell death by inhibiting the activity of endogenous cysteine proteases (Solomon et al., 1999, Yamada et al., 2000, Lampl et al., 2010, Chu et al., 2011, Martínez et al., 2012). Tajima et al. (2011) isolated a cysteine protease/inhibitor complex from spinach leaves and found that the corresponding genes were co-ordinately expressed in senescent leaves, and that removal of the inhibitor from the complex was necessary to activate protease activity. In Arabidopsis, the inhibitor AtSerpin1 acts to curb the protease activity of RD21 by preventing the ligation of peptides to the N-terminal of the acceptor protein (Lampl et al., 2010). In cauliflower, inhibition of protease activity has been linked to the activity of protease inhibitors that bind to and prevent maturation of proaleurains at the acidic pH present in vacuoles (Halls et al., 2006). Protease inhibitors have been assigned into classes: serine, cysteine, aspartate and metallocarboxy protease inhibitors (De Leo et al., 2002). However, functional analysis reveals that protease inhibitors may be active against multiple mechanistic classes of proteases (Habib and Fazili, 2007).

Phytocystatins are encoded by gene families, and member genes show different patterns of expression during plant development and defence (Martínez and Díaz, 2008). Expression is usually limited to specific organs or specific phases of development, such as fruit maturation and ripening (Ryan et al., 1998, Neuteboom et al., 2009), senescence (Huang et al., 2001, Sugawara et al., 2002, Tajima et al., 2011), PCD (Solomon et al., 1999), or defence against abiotic stresses (Gaddour et al., 2001, Van der Vyver et al., 2003, Diop et al., 2004, Zhang et al., 2008). Eight putative phytocystatins have been isolated from Brassica oleracea (cabbage) (Rawlings, 2010). A cDNA encoding one phytocystatin, BoCPI-1, has been isolated from B. oleracea var. italica (broccoli), and here we report on the functional characterisation of this protein. BoCPI-1 has inhibitory activity against cysteine proteases in vitro. We used genetic modification to alter its expression and show that the gene has a function in modulating postharvest protease activity, since overexpression delays the onset of postharvest senescence in the florets of broccoli heads.