Durian fruit pulp accumulates large quantities of GSH [5]. GSH is vital for plant cell homoeostasis [36] and serves as a storage form of Cys via incorporation with Glu and Gly in the γ-glutamyl cycle [37]. Cys is recycled from Cys-Gly hydrolysis. The latter dipeptide is a by-product of GSH breakdown via LAP. We hypothesise that this mechanism generates large amounts of precursors of the fruit-ripening phytohormone ethylene as well as considerable quantities of sulphur-volatile compounds via methionine synthesis. Taken together, both pathways lead to durian fruit ripening and its associated malodour. Cys-Gly was also detected in durian fruit pulp [5]. For these reasons, the γ-glutamyl cycle must be activated in durian fruit ripening. Our aims, then, were to identify and characterise the DzLAPs possibly involved in the aforementioned biochemical processes. To date, there has been limited information on the participation of Cys-Gly dipeptidase in the γ-glutamyl cycle [13, 15].
LAPs are highly conserved metallopeptidases in animals, plants, and microorganisms [38]. Several LAPs have been identified in Arabidopsis [13] and durian (this work). Arabidopsis has three LAPs encoding AtLAP1–AtLAP3. In the durian cv. Musang King genome [10], DzLAP1_MK and DzLAP2_MK were found. Based on transcriptome data, we focused on DzLAP1_MK as it was highly expressed in the durian fruit pulp (Supplementary Fig. S1) where numerous sulphur compounds such as GSH and γ-EC accumulate [5]. DzLAP1_MK showed 99.3% identity with DzLAP1 in the Chanee durian cultivar. The latter was the only isoform detected in our in-house RNA-seq data. The primary protein sequence analysis (Fig. 2) confirmed that DzLAP1 is a LAP-N because it contains all the conserved substrate binding/catalytic- and metal ion-binding residues responsible for the mechanism of this enzyme.
Postharvest DzLAP1 expression analyses showed upregulation at the midripe stage. Thus, DzLAP1 may hydrolyse Cys-Gly to Cys and Gly which results in the strong malodour associated with durian pulp during ripening. The relative DzLAP1 expression levels were similar for both Chanee and Monthong at each ripening stage (Fig. 3a). However, the Cys-Gly content in Chanee was significantly higher than that in Monthong (p < 0.01) [5]. Therefore, DzLAP1 is neither cultivar-dependent nor the key gene accounting for the relative differences in odour intensity between Chanee and Monthong. As the competitive ethylene inhibitor 1-MCP significantly repressed DzLAP1 during postharvest ripening (p < 0.05) (Fig. 3b), DzLAP1 must play an important role in durian fruit ripening. These observations imply for the first time that LAP1 is associated with the early stages of ripening. In contrast, the LAP-Ns in other plants are constitutively expressed in all organs [32, 39]. To clarify LAP expression during fruit ripening, we compared their expression levels in tomato fruit. The LAP-A expression levels in tomato fruit at the breaker or ethylene-producing stages were slightly lower than those in mature green fruit (Supplementary Fig. S2a). Tomato LAP-N is constitutively expressed at all fruit developmental stages (Supplementary Fig. S2b). Thus, LAPs are not implicated in tomato fruit development or ripening.
An in vitro biochemical assay of rDzLAP1 disclosed that LAPs have pH optima in the weakly to moderately alkaline range (pH 8.0–11.0) and metal ion dependency [27, 40, 41]. Thus, DzLAP1 is a metalloenzyme. We also predicted the in vivo substrate(s) for DzLAP1. This enzyme showed apparently high catalytic efficiency (kcat/Km) for Cys-Gly (Km = 1.6 mM). Therefore, DzLAP1 is a Cys-Gly dipeptidase. The aforementioned Km closely resembled those for other Cys-Gly peptidases such as AtLAP1 [13], yeast Dug1p [15], and bacterial TdLAP [12]. All of these were ≤ 1.3 mM. Thus, the affinity of LAP for Cys-Gly is relevant to the range of millimolar physiological GSH concentrations in living cells [42, 43]. Durian is GSH-rich compared to many other fruits and vegetables [5, 44]. For this reason, the cellular GSH level in durian should be higher than the general physiological concentration. This finding shows that Cys-Gly dipeptidases have highly conserved activity among various species. As with degradable α-linked dipeptides, DzLAP1 could not hydrolyse γ-Glu-Cys and GSH (γ-Glu-Cys-Gly) (Table 1). Consequently, this enzyme might have specificity for the α-peptide bonds in many cellular proteins. Nevertheless, previous studies did not investigate the affinities of LAPs for γ-linked dipeptide substrates.
Subcellular protein localisation could help clarify the correlation between the native functions and the physiological substrates of the enzyme. DzLAP1 is a dual-targeting protein localised to the chloroplasts and the cytosol (Fig. 5) even though it harbours a plastidial transit peptide sequence. DzLAP1 transcripts might contain an alternative translation initiation codon for ribosomes that skip the first start codon [32] and/or form secondary RNA structures in the sequences surrounding the start codon [45]. Delta-2-isopentenyl pyrophosphate:tRNA isopentenyl transferase (MOD5) is encoded by a single gene, contains two translational initiation sites, and is located in the mitochondria, cytosol, and nuclei [46]. Plastidial DzLAP1 may perform important functions such as general protein recycling. The chloroplast is intimately involved in cellular metabolism. Thus, it might perceive and respond to various stressors [47]. Although a function of plastidial DzLAP1 was not clarified in this study, we anticipate its function as a molecular chaperone preventing certain negative effects such as misfolded protein accumulation. The latter function has been reported for AtLAPs [48]. In addition, the partial localisation of DzLAP1 observed in the chloroplasts strengthens its chaperone or protease activity in the specific suborganellar compartment. Similar observations have been reported for CRP-like protein (Clp) and filamentous temperature-sensitive protein (FtsH) that are the major conserved ATP-dependent proteases with chaperone and degradative functions compartmentalising in stroma and on thylakoid membranes, respectively [49]. Further functional experiments are needed to confirm its role in the plastids during ripening.
Cys-Gly has pro-oxidant activity. For this reason, its concentration must be regulated. Cys-Gly promotes the formation of reactive oxygen species such as hydrogen peroxide and superoxide anion in the presence of certain metal ions [50]. Thence, oxidation of several normally highly reduced thiol compounds including GSH is induced [51]. The cytosolic localisation and enzyme kinetic parameters of DzLAP1 suggest that it participates in Cys-Gly hydrolysis in the cytoplasmic γ-glutamyl cycle. In this way, DzLAP1 controls the cytosolic Cys-Gly levels. Moreover, Cys-Gly may be a substrate for cytosolic DzLAP1 in vivo and the enzyme effectively regulates its concentration. DzLAP1 expression and localisation in the cytosol where ethylene and sulphur volatiles accumulate might indicate the involvement of this enzyme in durian fruit ripening. The slightly alkaline pH optimum for DzLAP1 activity is consistent with the relatively basic cytoplasm [52] and chloroplast stroma [53, 54].
DzLAP1 shares certain important features of the LAP-N in Arabidopsis and Solanaceae including similar catalytic/substrate and metal ion-binding residues. However, the DzLAP1 expression pattern differed from those of the other genes. Durian has evolved to accumulate high levels of sulphur compounds that impart a very strong and unique odour to the fruit. Nevertheless, the levels of these thiols must be tightly controlled. Therefore, expression of the genes encoding sulphur-metabolising enzymes must be regulated in durian fruit ripening. The DzLAP1 promoter region may have been evolutionarily modified such that the optimal DzLAP1 content is produced to limit the amount of Cys-Gly and catalyse Cys recycling in order to the generation of sulphur volatiles and ethylene during fruit ripening. A similar finding was observed in the durian-specific gene expansion of methionine γ-lyase [10], a major enzyme responsible for sulphur volatile production in plants and microbes [55, 56]. A fruit ripening-associated isoform of this enzyme has been identified [10]. Taken together, we present a schematic diagram summarising the putative functions of DzLAP1 in the cytoplasm and chloroplasts of durian fruit pulp (Fig. 6).