Introduction The Bromeliaceae family is one of the
Introduction The Bromeliaceae family is one of the most morphologically and ecologically diverse flowering plant families native to the tropics and subtropics of the New World (Benzing, 2000). They comprise >3000 species and are traditionally divided into three subfamilies: Pitcairnioideae, Bromelioideae, and Tillandsioideae (Givnish et al., 2011; Silvestro et al., 2014). Tillandsioideae is the third largest genus endemic to Brazil, with the Atlantic Forest being its primary center of NHS Biotin (Barfuss et al., 2016). All species of this subfamily are epiphytic and dependent of their leaves to absorb nutrients which are dissolved in rain, the stemflow, and throughfall water, while roots act primarily as holdfasts. In addition, decomposed vegetable and animal debris accumulated into the tank can be rich in nutrients that are absorbed by foliar trichomes (Martin, 1994). Vriesea carinata is an epiphytic bromeliad that belongs to Tillandsioideae and considered as tank-type (epiphytic) bromeliad which allows the trichomes at the leaf bases to be in contact with water and nutrients for a prolonged time (Benzing, 2000). It is found in humid and well-preserved sites (Martinelli et al., 2008), and has a wide distribution in the Brazilian Atlantic Forest. It has flowers of various shades of red with each rosette producing a single inflorescence (da Costa et al., 2014). For this, it is often used as an ornamental plant, which causes them to be illegally extracted. The increased extractivism of this species, linked to the fragmentation of the Atlantic Forest, has caused concern with respect to the conservation of V. carinata, as well as many other bromeliad species (Zanella et al., 2012). In plants, the proteolysis is a complex process involving many enzymes in various cellular compartments, with cysteine proteases (CysPs) playing an essential role. They are involved in protein maturation, degradation, and protein rebuilt in response to different external stimuli and they also play a house-keeping function to remove abnormal, misfolded proteins. They can be grouped in 10 clans subdivided into 15 families (Rawlings et al., 2014). Among these, the CD clan is comprised by legumains and metacaspases. Legumains (family C13) are lysosomal/vacuolar CysPs, also called Vacuolar Processing Enzymes (VPEs), and were first described in plants where they contribute to the processing and maturation of seed storage proteins (Kembhavi et al., 1993). Metacaspases (family C14) are plant CysPs that recognize residues of arginine (R) or Lysine (K) at the substrate cleavage site instead of Aspartic Acid (D) residues used in animal caspases. They are mainly involved in the regulation of programmed cell death (Bozhkov et al., 2005; Vercammen et al., 2004). The CF clan has pyroglutamyl-peptidase I as its unique peptidase member. They hydrolytically remove the pyroglutamate (pGlu) residue from the amino terminus of pGlu peptides and proteins and have been observed in the tissues of mammals, birds, fish, plants, and bacteria (Szewczuk and Kwiatkowska, 1970; Szewczuk and Mulczyk, 1969). In the CA clan, we have calpains and papain-like CysPs. Plant genomes contain only one calpain (family C02), known as phytocalpain, which is essential for plant epidermis development (Lid et al., 2002; Margis and Margis-Pinheiro, 2003). Papain-like cysteine proteases (family C1) (PLCPs) are very stable enzymes and are often found in proteolytically harsh environments such as the apoplast, vacuole, and lysosomes (Richau et al., 2012). PLCPs also play key roles in programmed cell death (PCD) in the development process, as well as that induced by external stimulus, as previously mentioned CysPs (van der Hoorn, 2008). The PLCP family consists of papain, chymopapain, caricain, bromelain, actinidin, ficin, aleurain, etc. Research on CysPs has mainly focused on Bromelioideae family, of which pineapple (Ananas comosus) is the most studied species, since they contain substances and vitamins that are beneficial for human health. Therefore, bromelain is the collective name of related CysPs found in species belonging to the Bromeliaceae family.