Several plant species, including cotton, ficus, peanut, and orange, contain genes encoding for papain-like (PLCP) cysteine proteases of the C1A subfamily (Zhang et al. 2019; Zhai et al. 2021; Zhang et al. 2023; Li et al. 2023). Species have different numbers of C1A protease genes. The best-studied genome in the Bromeliaceae family is A. comosus, which has 71 protease genes, four of which have been related to recognized activities and named as true bromelains (Yow et al. 2023). In our study, we found six PrBLCP genes in P. raimondii. The intron-exon structure, motif and conserved domains of all genes were comparable (tables S1 and S6). These traits have been seen in C1A genes across other species exhibiting analogous gene architectures and functional domains (Zhai et al. 2021; Li et al. 2023). At the nucleotide level, all sequences found in P. raimondi had more than 80% similarity with the genome of A. comosus, suggesting that these proteins are highly conserved (table S5).
Overexpression of ARE, LTR, MBS, STRE, MYC, Unnamed 4, AE-box, AT1-motif, ATC-motif, Box4, Gap-box, GATA-motif, G-box, GT1-motif, I-box, MRE, Sp1, and TCT-motif in the promoters of PrBLCPs genes in P. raimondii suggested a role for these proteases in stress responsiveness. Different variables affect gene expression via cis-regulatory regions. These areas have enhancers, repressors, and transcription factor binding sites for spatiotemporal gene expression (Marand et al. 2023).
The role of cysteine proteases in plant stress response has received a lot of attention. Response elements to hormones, light, drought, low temperature, and wounding have been discovered in A. comosus genes related to stress response such as HSF (Wang et al. 2021), plant-pathogen interaction genes such (Zhou et al. 2023) and genes regulating developmental processes such as AP2/ERF (Zhang et al. 2021). It has been suggested that under stressful circumstances, multiple regulatory mechanisms interact. DRE (dehydration-responsive element) cis-elements bind DREB proteins, causing dehydration stress response proteins and transcription factors to be activated. DREB1 (cold-inducible) gene promoters, on the other hand, overexpress hormone-responsive motifs (auxin, GA, ethylene, ABA, Me-JA) (Srivasta et al. 2010). ABRE (abscisic acid-responsive) and light-responsive elements, on the other hand, are overexpressed in DREB2 (drought-inducible) promoters. W-box and WUN motif elements are associated with wound response and have been discovered in the promoters of DREB genes (Ain-Ali et al. 2021). Light-responsive elements, such as Box 4 and G-box, have been found in the promoters of genes that are drought, salinity, cold, or heat-responsive (Shariatipour and Heidari 2020). The findings of this research suggest that the expression of PrBLCPs genes may be associated with the capacity of Puya species to adapt to arid conditions, such as the steep slopes and rocky outcrops that serve as their natural environment.
Although not all mechanisms are understood, bromelains are known to participate in the hypersensitivity response and a type of programmed cell death associated with this response (Salguero-Linares and Coll 2019). They found that Candidatus liberibacter infection produced CsPLCPs in Citrus sinensis (Li et al. 2023). Zhao et al. (2023) demonstrated that cytokinins, ethylene, and methyl jasmonate all promoted PLCP gene expression in grapes, which in turn-controlled resistance to Phytophthora capsici via hormonal signaling pathways. In fig, light positively regulates PLCP gene expression and herbivory control is linked to high PLCP gene expression and ficin accumulation in the inflorescence, receptacle, and latex (Zhai et al. 2021). PrBLCPs expression is linked to pathogen and herbivore defense genes in P. raimondii. These findings aid species management and phytosanitary control, enabling conservation measures to conserve natural populations.
MEROS classifies papain-like cysteine proteases (subfamily C1A) as inactive proenzymes with an inhibitory domain I29 and a peptidase domain C1. In these enzymes, the inhibitory domain I29 acts as a propeptide of the inactive zymogen, preventing substrate access to the active site. It also helps enzyme folding and transport (Wiederanders 2003). These domains and catalytic amino acids are conserved in this subfamily. All PrBLCPs found in this work had a propeptide with the inhibitory domain I29 and peptidase domain C1. PrBLCPs, like other cysteine proteases, include the heptapeptide GXNXFXD, which is highly conserved in the propeptide and helps these enzymes fold correctly, according to Ramli et al. (2018) the D residue of this motif participates in the correct folding of these enzymes.
The core structure of P. raimondii PrBLCPs is quite like fruit bromelain, as shown by multiple alignment and functional domain analysis. Physicochemical properties suggest they are more like fruit bromelain than stem and ananain (table S7). Purified stem bromelain has 212 amino acids, glycosylation at residue N117 and a molecular mass of 23.40-35.73 kDa and pI 9.55 (Ritonja et al. 1989). Fruit bromelain contains 351 amino acids, 25–31.00 kDa molecular mass, no glycosylation and pI 4.6 (Yamada et al. 1976). Ananain, on the other hand, is a non-glycosylated 216-residue sequence with a theoretical mass of 23,464 kDa (Lee et al. 1997). Another distinguishing characteristic found in PrBLCP sequences is an amino acid insertion (GTKYW) between positions 170–174 (ananain numbering), which is comparable to ananain. This area is found in fruit bromelain as well as other cysteine proteases including chymopapin and actinidin, but not in stem bromelain or papain (Lee et al. 1997). Ananain crystal structure investigations revealed that this insert creates the -chain beginning and operates by stabilizing the D167-S168-S169 loop structure (Yongqing et al. 2019). Although these proteases are quite similar, with substantial similarity in their main sequences, they vary in substrate selectivity, inhibitory characteristics and immune response (Azarkan et al. 2020).
The C1 peptidase domain is a monomer of 150 to 200 amino acids that constitute two domains structurally, the L domain with α helices and the R domain with β antiparallel sheet structure. Residues C26 and H158, positioned in the gap between the R and L domains, include the unique catalytic dyad of C1A subfamily enzymes. The R domain residue N179 and the L domain residue Q20 help to generate the oxanionic cavity and make hydrogen bonds with the protonated side chain of residues H158 to orient the imidazole ring (Azarkan et al. 2020). These active sites were found in PrBLCP2, PrBLCP3, PrBLCP4, and PrBLCP5, however, PROSITE did not detect C26 as an active site in PrBLCP1 (Fig. 4). This might be explained by the replacement of G24 by S24, which results in two consecutive serine residues (S24-S25) upstream of C26. The SH group of C works as a nucleophile by giving a pair of electrons in the catalytic process, but two OH groups so near to the S groups might substitute the SH function and prevent PROSITE from recognizing residue C as an active site (Fig. 3). The potential folding of PrBLCP1 shows residue C26 orientated toward the oxanionic cavity, as in other bromelain-like, therefore enzymatic experiments are required to determine whether C mediates proteolytic action. The estimated structural model for PrBLCP1, PrBLCP2, and PrBLCP3 shows a typical bromelain folding that is comparable to O23791 (fruit bromelain) (Figs. 6 and 7).
Phylogenetic relations between P. raimondii putative bromelain-like cysteine proteases with A. comosus bromelains placed the PrBLCPs in two subclades, however the distances are minor, possibly due to recent evolutionary changes (Fig. 5). PrBLCP1, PrBLCP2, and PrBLCP3 grouped around the actual A. comosus bromelains discovered by Yow et al. (2023) (ACMD2v2v2_05.00101, 05.00102, 05.02951, and.02955). No difference in stem or fruit expression of these genomic sequences codes for commercial bromelains with recognized activities. PrBLCP4, PrBLCP5, and PrBLCP6 were connected to the most ancestral A. comosus bromelain orthogroup members (ACMD2v2_16.26904, 16.26905 and 1626906). The distances between the two subclades in the phylogram imply two major bromelain clusters, presumably from one orthologous gene family. These findings support the recent and fast divergence of these species and their high gene family overlap (Liu et al. 2021). The great conservation of these genes and the structural properties of the proteases they express imply that bromelains are crucial to Puya species evolution and adaption to severe conditions.
The C1A protease family members have crucial roles in responding to abiotic stressors, such as regulating cell death programs and protecting against herbivory and pathogen infections. Currently, there is a lack of characterization research on these enzymes specifically in the species Puya. The findings of this study, together with its application to other species within the same genus, might serve as a valuable addition to the understanding of the regulatory mechanisms of these proteases in plants. Furthermore, the use of cysteine proteases in fields such as agriculture, biotechnology, and health may have implications for the preservation of plants belonging to the Bromeliaceae family amidst the ongoing climate change.