Detoxification-related gene expression accompanies anhydrobiosis in the foliar nematode (Aphelenchoides fragariae)

Upregulation of detoxification-related gene and gene sets

In total, 2,083 and 953 genes were significantly up- and downregulated, respectively, in desiccated nematodes (Fig. 2A). Of the 100 annotated genes with the largest positive fold-changes (box, Fig. 2A), more than one third encoded putative detoxification-related proteins (Fig. 2D). These included numerous Phase I and II detoxification enzymes: CYPs, SDRs, UGTs, and GSTs. Also among the top hundred upregulated genes were a pgp-14-like multi-drug resistance protein (MRP/PGP), an FAD-dependent monooxygenase, and multiple nuclear hormone receptors (NHRs), all of which have been implicated in the cellular detoxification program (Fig. 2; Lindblom and Dodd, 2006; Hodge and Tracy, 2010; Adhikari and Adams, 2011; Hoffmann and Partridge, 2015; Harder, 2016).

Figure 2:

Across the transcriptome as a whole, large percentages of CYPs (45%), SDRs (32%), UGTs (55%), NHRs (30%), and MRP/PGPs (32%) were upregulated in response to desiccation (Fig. 2B). Only the GSTs did not respond strongly as a group: 28 GST genes were assembled: 5 (18%) were significantly upregulated and three were downregulated.

Results of gene set enrichment analysis were consistent with wholesale induction of detoxification-related genes. Of the top 15 gene sets enriched during desiccation, 5 were dominated by detoxification-related genes. The majority of leading-edge genes in each set (i.e. those that contributed to the enrichment signal) were again CYPs, UGTs, NHRs, and MRP/PGPs. Gene sets associated with other detoxification-related GO terms were also significantly enriched (Fig. 2C): xenobiotic transport (GO:0042908), xenobiotic transporting ATPase (GO:0008559), steroid hormone receptor (GO:0003707), and glucuronosyltransferase (GO:0015020). The leading-edge genes of the former two sets were made up entirely of MRP/PGP genes. The leading-edge genes for GO:0003707 were entirely NHRs, while those of GO:0015020 were primarily UGTs.

CYPs and SDRs are canonical enzymes of Phase I detoxification that add reactive functional groups to a wide variety of endogenous and exogenous compounds. Both are large gene families: 86 CYPs and 68 SDRs have been documented in C. elegans, and 80 CYPs and 84 SDRs were expressed here in A. fragariae (Lindblom and Dodd, 2006). CYPs primarily hydroxylate lipophilic substrates, including endo- and xenobiotics, steroids, and fatty acids. SDRs also activate lipophilic substrates by catalyzing the reduction of carbonyl groups in aldehydes and ketones. A number of strongly upregulated SDRs in A. fragariae were similar to the uncharacterized C. elegans’ short chain dehydrogenase dhs-27, an ortholog of human HSDL1 (hydroxysteroid dehydrogenase like 1). The reactions catalyzed by CYPs and SDRs are energetically expensive, consuming NADH/NADPH during substrate functionalization (Gems and McElwee, 2005).

Functionalized substrates are further modified by the Phase II reactions catalyzed by UGTs and GSTs. These reactions typically involve the addition of side groups that increase the substrate’s solubility in preparation for excretion. Like CYPs, UGTs act on numerous small lipophilic compounds: xenobiotics, endogenous waste metabolites, steroids, and fatty acids. GSTs participate in the modification and detoxification of substrates by multiple mechanisms, including the addition glutathione to an electrophilic substrate and the direct binding of toxic substrates. These are also expensive reactions: each UGT glucuronidation reaction requires one molecule of glucose, while each GST transferase reaction uses one molecule of reduced glutathione (Gems and McElwee, 2005; Lindblom and Dodd, 2006). Following enzymatic modification by Phase I and II enzymes, toxins and waste metabolites are excreted from the cell by membrane transporters, mainly members of the ATP-binding cassette (ABC) family of efflux pumps (Lindblom and Dodd 2006). Among this large protein family, MRP and PGP transporters have been most extensively characterized for their role in detoxification (Choi, 2005; Hoffmann and Partridge, 2015; Harder, 2016).

NHRs are a very large class of transcription factors (over 280 in C. elegans). In most cases, NHRs bind lipophilic hormones, e.g. steroids, retinoids, and fatty acids, and also regulate gene expression through the integration of endogenous and exogenous signals. Specific NHRs, such as NHR-8 and NHR-48 in C. elegans and DHR96 in Drosophila, regulate xenobiotic detoxification network in response to environmental conditions (Hoffmann and Partridge, 2015). A gene (DN12692_c0_g8) in our data set that is homologous to nhr-48 was upregulated despite its low expression. Additionally, DAF-12 (C. elegans), NHR-8, and DHR96 are involved in metabolism of steroid hormone, cholesterol, and triacylgylceride, which are related to healthy aging (King-Jones et al., 2006; Bujold et al., 2010; Horner et al., 2009; Antebi, 2013; Magner et al., 2013; Wang et al., 2015). Desiccated nematodes undergo extended life span, and it is critical to maintain health during such process. Our data have shown that genes that are key to xenobiotic detoxification network were activated in desiccated nematodes, suggesting the connection between desiccation and healthy aging via detoxification.

The broad induction of detoxification-related genes was the single most striking pattern to emerge from the desiccation-related transcriptome. This result is intriguing, as it parallels the broad induction of detoxification-related gene expression that has been reported in C. elegans’ stress-tolerant, growth-arrested dauer larvae and in the long-lived C. elegans daf-2 mutant (Gems and McElwee, 2005; Erkut et al., 2013). Both dauers and long-lived mutants upregulate CYPs, SDRs, UGTs and GSTs (McElwee et al., 2004). Evidence suggests that induction of the detoxification program may occur broadly in response to dehydration: several previous studies of anhydrobiotic organisms have noted upregulation of detoxification-related genes. Desiccation-related ESTs from the Antarctic nematode, Plectus murrayi, included a GST, an NHR, an aldehyde dehydrogenase, and several ABC transporters. Among the most highly represented KEGG pathways in the EST library was “xenobiotic and bio-degradation” (Adhikari et al., 2009; Adhikari and Adams, 2011). A CYP was one of 15 dehydration-responsive genes identified by suppressive subtractive hybridization in the Antarctic midge, Belgica antarctica (Lopez-Martinez et al., 2009), and multiple CYPs and GSTs were induced by dehydration in the dog tick, Dermacentor variabilis.

This raises the question of what, exactly, is being detoxified in stress tolerant and/or long-lived organisms. Anhydrobitic A. fragariae and C. elegans dauers do not feed, it is therefore unlikely that detoxification reactions are required to process exogenous xenobiotic toxins. It has been suggested that these detoxification reactions are used to eliminate accumulated endogenous lipophilic wastes, the products of stochastic errors in metabolism or stress-related damage (Gems and McElwee, 2005). Alternately, detoxification enzymes are also involved in hormone and fatty acid metabolism, and these functions may be the principle means by which they influence stress tolerance, development and longevity (Zimniak, 2008; Hodges and Minich, 2015). These potential roles—clearing of endogenous lipophilic waste, hormonal regulation of development, and control of fatty acid metabolism—are not mutually exclusive. All may play a part in survival and longevity. Detoxification reactions are energetically expensive, and the fact that detoxification-related gene expression is prioritized under non-feeding, stressful conditions suggests detoxification, while costly, is vital to survival during and/or after release from anhydrobiosis.

Heat shock proteins, chaperones, and the unfolded protein response

Desiccation can cause protein misfolding, damage, and aggregation (Tapia et al., 2015). Indeed, such damaged proteins may be important substrates for the detoxication enzymes highlighted above, such as CYP, UGT, SDR, and NHR. Multiple genes encoding molecular chaperones and components of the unfolded protein response were induced by desiccation in A. fragariae (Fig. 3). These molecular chaperones support proper protein folding and reduce protein aggregation by both ATP-dependent and ATP-independent mechanisms (Hartl et al., 2011; Basha et al., 2012).

Figure 3:

Of the 98 heat shock protein genes assembled from A. fragariae, 11 were upregulated and three were downregulated (Fig. 3A). Upregulated genes encoded chaperones from the hsp20, hsp40, and hsp70 families. It should be noted that many hsp genes were expressed at high levels under both desiccated and control conditions. For example, four hsp90 genes and three hsp70 genes were expressed at levels greater than 1000 FPKM under both conditions (Supplementary File 3, https://doi.org/10.5061/dryad.8pk0p2njc). Also significantly upregulated by desiccation were the molecular chaperones bag-2 and calnexin (Fig. 3A). The latter is an endoplasmic reticulum (ER) chaperone that functions in the unfolded protein response (Takayama et al., 1999; Lee et al., 2005).

Desiccated A. fragariae also showed upregulation of additional unfolded protein response genes and enriched expression of related gene sets (Fig. 3B). Upregulated genes included UGGT (UDP-glucose glycoprotein glucosyltransferase), which adds glucose residues to misfolded glycoproteins; alpha-1,2-mannosidase, which removes mannose residues from misfolded proteins; and alpha-mannosidase EDEM3, which trims mannose residues and directs the trimmed proteins to the ER-associated degradation pathway (Sousa and Parodi, 1995; Hosokawa et al., 2001; Hirao et al., 2006). Finally, pek-1, whose encoded protein globally suppresses translation during ER lumen stress, was also induced by desiccation (Richardson et al., 2011).

Intrinsically disordered proteins

Another means by which dehydrating organisms may prevent catastrophic damage to proteins and membranes is through the production of strongly hydrophilic, IDPs. These proteins have been documented in anhydrobiotic species from multiple kingdoms of life. Often classified as late embryogenesis abundant (LEA) proteins, IDPs are thought to stabilize proteins, membranes, and organelles during desiccation (Hand et al., 2011). Recently, novel desiccation-induced IDPs with no homology to other known proteins were identified in tardigrades and shown to mediate desiccation tolerance (Boothby et al., 2017).

A subset of strongly upregulated A. fragariae transcripts appeared to code for IDPs. The putative A. fragariae IDP transcripts received no annotations in the Blast2GO pipeline despite possessing open reading frames longer than 200 bp and substantial read support. Their fold-changes and absolute FPKM values were high, and some received significant blastx hits to the LEA Protein database. Most contained tandem repeats of short amino acid motifs, a common feature of IDPs (Jorda et al., 2010). All were enriched in disorder-promoting amino acid residues such as glycine, serine, lysine, and glutamine (Uversky, 2019). In total, 14 such transcripts are presented in Table 1, with information on their length, predicted disorder, hydropathy, and blastx hits to known LEA proteins.