Cladonia arbuscular holobiont systems produce enzymes that degrade plant and microbial biopolymers in an alpine shrub-heath.

Cladonia arbuscular holobiont systems produce enzymes that degrade plant and microbial biopolymers in an alpine shrub-heath. | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 26 March 2025 V1 Latest version Share on Cladonia arbuscular holobiont systems produce enzymes that degrade plant and microbial biopolymers in an alpine shrub-heath. Authors : Robert Mills 0000-0002-4020-5129 [email protected] , Josh Thurston , and Jake Spong 0000-0002-0815-0316 Authors Info & Affiliations https://doi.org/10.22541/au.174298689.98349959/v1 228 views 108 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract not-yet-known not-yet-known not-yet-known unknown Lichens are a conspicuous part of many ecosystems, especially arctic-alpine heaths and drylands, but their direct role in ecosystem functioning lags behind knowledge on vascular plants and other cryptogams. Lichen holobiont systems can produce enzymes In particular, the propensity for lichens to produce enzymes to degrade organic matter is poorly understood. We quantified the production of enzymes degrading microbial and plant-litter biopolymers, and tested if these activities were altered by proximity to typical alpine dwarf shrubs. We used a gentle extraction to detect enzymes that may be mobile in the environment and thus contribute to soil enzyme pools. We found that Cladonia arbuscular produces hydrolases that can degrade a range of microbial and plant litter biopolymers, but crucially that the production of plane-litter enzymes can be augmented by the presence of dwarf shrubs. Together this highlights new biogeochemical pathways in lichen system, where they may play a role in complete degradation of plant litter outside of the soil, contribute enzymes and partially-degraded organic matter to soil, and that the magnitude of these effects is a function of lichen:vascular plant interactions. Further work is urgently required to seek generality in these findings, identify the biotic and abiotic constraints to lichen enzyme production, and provide parameters to improve biogeochemical models. not-yet-known not-yet-known not-yet-known unknown Abstract Lichens are a conspicuous part of many ecosystems, especially arctic-alpine heaths and drylands, but their direct role in ecosystem functioning lags behind knowledge on vascular plants and other cryptogams. Lichen holobiont systems can produce enzymes In particular, the propensity for lichens to produce enzymes to degrade organic matter is poorly understood. We quantified the production of enzymes degrading microbial and plant-litter biopolymers, and tested if these activities were altered by proximity to typical alpine dwarf shrubs. We used a gentle extraction to detect enzymes that may be mobile in the environment and thus contribute to soil enzyme pools. We found that Cladonia arbuscular produces hydrolases that can degrade a range of microbial and plant litter biopolymers, but crucially that the production of plane-litter enzymes can be augmented by the presence of dwarf shrubs. Together this highlights new biogeochemical pathways in lichen system, where they may play a role in complete degradation of plant litter outside of the soil, contribute enzymes and partially-degraded organic matter to soil, and that the magnitude of these effects is a function of lichen:vascular plant interactions. Further work is urgently required to seek generality in these findings, identify the biotic and abiotic constraints to lichen enzyme production, and provide parameters to improve biogeochemical models. not-yet-known not-yet-known not-yet-known unknown Keywords Hydrolase, lichen, decomposition, alpine, extracellular, glucosidase, biogeochemistry, heath. not-yet-known not-yet-known not-yet-known unknown Introduction Extracellular enzymes (EEs) catalyse the depolymerisation of biopolymers, and thus are the rate-limiting step for decomposition of organic matter (Burns et al., 2013). It is generally assumed that enzymes degrading plant-litter are found predominantly in soils and on senesced plant litter, and consequently, models generally concentrate on soils when describing enzyme-mediated processes (Wang et al., 2022). In contrast to the focus on ‘in soil’ processes, little attention has been placed on lichens and other cryptogams as a source of plant-degrading enzymes, or as a location for litter decomposition, with the majority of focus on indirect effects (Cornelissen et al., 2007). Lichens and their microbiomes produce EEs, including oxidative enzymes (Beckett et al., 2003) and some hydrolases (Miralles et al., 2012), but the focus on hydrolases has been mainly around phosphorus (Higgins and Crittenden, 2015). There has been little emphasis thus far on the role lichens play in direct litter degradation, or the production and mobility of enzymes that degrade the most abundant plant (cellulose, hemicellulose) and microbial (chitin) biopolymers. The majority of lichen-litter interactions focus on the microclimate-modifying effects of lichen cover (van Zuijlen et al., 2020) and on the activity of soil enzymes (Sedia and Ehrenfeld, 2006), as well as the role lichen leachates may play in altering soil microbial communities (Dostos et al., 2022)). Terricolous lichens are common in arctic and alpine environments, and in particular, the Cladonia genus can become locally dominant at scales of decimetres to tens of metres in northern arctic-alpine environments and boreal forest ecosystems, which raises questions into the role they play in wider biogeochemical cycling. The interplay of Cladonia species such as arbuscula, rangiferina, stellaris, and portentosa, with dwarf shrubs is a distinctive feature of these communities, yet the majority of functional ecological study focuses on the role of vascular plants. This hinders understanding and predictions of change, especially crucial at a time when lichens may be the more resistant and resilient facets of natural systems under rapidly-changing climate (Mayo de la Iglesia et al., 2024). To predict the impacts of change, and develop meaningful biogeochemical models for lichen systems, a deeper understanding of their role in biogeochemistry is much needed. Collections of aboveground biomass from heaths featuring Cladonia often show strong physical attachment of Cladonia thalli to senesced leaves of dwarf shrubs (Fig. 1), suggesting the Cladonia holobiont system is actively degrading the adhered litter, which is an unexplored aspect of their functional ecology. If this is the case, we expect to see higher rates of enzyme production when Cladonia are found in close contact with shrubs. There may, due to litter-quality differences, be species-specific differences in Cladonia enzyme production depending on neighbouring dwarf shrub species. To explore these broad ideas, we asked: 1. Do Cladonia holobiont systems produce hydrolases to degrade dominant plant and microbial biopolymers?; 2. Does the species identity of dwarf shrubs growing with Cladonia alter the activity of EEs? Materials and methods not-yet-known not-yet-known not-yet-known unknown Site description and sampling Samples were collected from the Geal Charn research site, Cairngorms, UK 57°05’28”N 3°50’58”W in July 2024. The study area was a 50*100 m polygon at 800m a.s.l. on a south-facing slope typified by the Calluna-Cladonia alpine shrub heath with dominant (determined by spatial cover) shrubs Calluna vulgaris, Empetrum nigrum, Arctostaphylos urva-ursi, with infrequent cover from Vaccinium vitis-idea and Vaccinium myrtillus . Cladonia arbuscula is the dominant lichen species, within a highly biodiverse cryptogam community. We defined four scenarios to test the effect of shrub partnering on Cladonia EEs, with one scenario representing complete local dominance of Cladonia (Cladonia arbuscula present with no vascular plants within a 20 cm radius), and three scenarios reflecting shrub co-occurrence, where the target shrub (Calluna vulgaris, Empetrum nigrum, Arctostaphylos urva-ursi ) occupied 50% of a 10cm radius circle with Cladonia arbuscula as the only partner. We identified 12 sample points for each scenario, and sampled and bulked thalli from three 5cm-diameter sub-clumps where the physical interaction of thalli and shrub leaves/stems was maximal. Samples were stored fresh in open plastic bags in a cool box, then returned to York 48 hours later, kept at 4°C in the dark for two days, before sample processing. Sample processing and enzyme activity Thalli were cleaned of litter fragments and soil using tweezers. The distal 4 cm of thalli were cut and retained, to standardise, and reduce contamination from soil enzymes adhered to basal parts. Enzymes were extracted according to Jassey et al, (2012), except our samples were inverted on an end-over-end shaker for 2 hrs at 20 rpm to reduce extraction energy, and more closely reflect enzymes removed by intense rain events. We focussed on four EEs to address our research questions. Two, acid phosphatase (PHO) and N-acetyl glucosaminidase (NAG), are expected to be unaffected by shrub presence as these EEs target P acquisition from membrane-bound phosphate and microbial/invertebrate biopolymers (chitin/peptidoglycan) respectively, and the activity of these enzymes are not expected to be proportional to plant litter. Two plant-litter degrading hydrolases; β-glucosidase (BG), which degrades cellulose, and xylosidase (XYL), which degrades hemicellulose, were selected as they control degradation of the two dominant plant biopolymers, and thus ought to be a good indicator of investment in decomposition of plant litter at the thallus surface. Determination parameters followed those described in Kuttim et al (2017). not-yet-known not-yet-known not-yet-known unknown Data analysis Enzymatic activity rates were compared amongst scenarios using ANOVA after inspection of assumptions, with log-transformations applied when needed to satisfy normality, and using Tukey’s honest significant difference post-hoc test for pairwise comparisons. All analysis was carried out in R 4.3.2 ‘eye holes’. Results We detected activity of all four enzymes in all 48 samples, meaning hydrolases capable of degrading microbial necromass and plant litter biopolymers are present, active, and easily leached from the Cladonia arbuscula holobiont system. Activity was generally highest for acid phosphatase, and across all shrub contexts the standard deviation was large. The effect of shrubs on Cladonia activity was enzyme and species specific (Fig. 2). Although mean values for PHO activity were higher in all shrub-associated Cladonia , there was no overall effect of shrub species, nor any pairwise differences. This was also the case for NAG. There was an overall effect of shrub on XYL activity (F (3,43) = 12.8, P2 times greater when co-occurring with Arctostaphylos than Cladonia alone, or when with Calluna or Empetrum . For β-glucosidase, shrub species had an overall effect (F (3,41) = 5.8, P=0.002), and activity was higher when Cladonia co-occurred with all shrubs than when alone, whilst amongst-shrub comparisons were not significantly different from each other (above 0.05 level). Taken together, in general, the presence of shrubs increased the activity of plant-litter degradation enzymes, but not chitin-degrading or phosphate-ester degradation. not-yet-known not-yet-known not-yet-known unknown Discussion Cladonia arbuscula holobiont systems produce hydrolases capable of degrading plant litter, and this production is altered by the presence of dwarf shrubs, with species-specific effects. In some cases, mean effects were noticeable, but statistical comparisons were above the 5% level and variance was high across all samples, despite our high replication (n=12). This suggests there are likely to be some within-thallus effects (i.e. stronger EE response when in direct contact with shrub litter) and variation in the holobiont microbiome, which should be explored by higher-precision sampling at the thallus-shrub interface and comparative metabarcoding of thallus microbiomes. Wider breadth in assayed enzymes would improve the clarity of the observations, and given EE production can vary seasonally (Puissant et al., 2018), temporal measurements would better link to litter production and abiotic constraints. Depolymerisation of cellulose and hemicellulose in Cladonia thalli has not been previously reported, and that shrub-presence enhances this is compelling evidence that community structure can augment key biogeochemical pathways of C cycling outside of the soil, and may be manifest in other enzymes catalysing C and nutrient dynamics. The ease with which we were able to extract meaningful quantities of EEs from Cladonia thalli suggests the transfer of enzymes from the lichen canopy to the soil is occurring, and is likely a feature of precipitation events and fragmentation of lichen tissue which enters the soil litter pool. This ‘enzyme rain’ entering the soil from cryptogam surfaces may contribute to the total enzyme pool of soil, and along with other dissolved and particulate material sourced from the cryptogam surface, could be broadly analogous to inputs from vascular plants via rhizodeposition in terms of stimulating or limiting soil microbial processes. However, most focus so far has been on the potential antimicrobial roles of these leachates (Rankovic et al., 2010; Stark et al., 2007). Clearly there is a need to quantify in situ inputs of enzymes to soil, constraints to the magnitude and frequency of these inputs, as well as the proportion of the total soil enzyme pool which is derived exogenously. Further, there is a need to understand the persistence of these enzymes in soil, and whether any physiological differences between those produced on cryptogam surfaces and those produced in soil confer any flexibility in activity (e.g. thermal sensitivity/tolerance, substrate-enzyme affinity kinetics) or stability/turnover time (Schimel et al., 2017). This is especially crucial given enzyme parameters are identified as a key uncertainty in current biogeochemical models (Saifuddin et al., 2021). These uncertainties tend to be largest at the end-members of hydrological gradients (Sierra et al., 2015), and these hydrological end-members also tend to be more cryptogam-dominant (e.g. biocrust drylands, lichen-rich wet heaths, moss-dominated bogs), meaning cryptogam processes may contribute to this uncertainty. This is especially important under climate change, where hydrological shifts could alter the abundance of cryptogams (Schwager et al., 2024). Our observations are at the holobiont level, and we recognise that these whole-thallus extractable enzyme pools will be derived from both Cladonia and its microbiome. A key research challenge will be to probe the wider enzyme-production capability of the system and the organisms behind these activities. It is now imperative to track the mobility and persistence of lichen-derived enzymes, and those derived from other surfaces such as the phyllosphere of other cryptogams and vascular plants. The next iteration of biogeochemical models will benefit from these new parameters, especially given the abiotic controls over surface enzyme production may contrast considerably with those found in soil. The community of ecosystem ecologists is well-placed to rapidly assess the generality of our observations and the magnitude of cryptogam-derived enzyme contributions to whole ecosystem functioning, and that this approach can be strengthened through insight into the active microbiome community, and parsing out the contributors to these EE pools using ‘omics and simultaneous enzymology assays. not-yet-known not-yet-known not-yet-known unknown Conclusion Cladonia arbuscula holobiont systems produce extracellular enzymes to degrade a range of biopolymers, and those related to plant litter are augmented in some cases by the presence of dwarf shrubs. These enzymes are easily extracted, may be mobile in the cryptogam-soil system, and are overlooked as a potential source of soil enzymatic activity. Revealing the breadth of this pattern amongst the diverse suite of enzymes produced in the holobiont system, and the generality of this amongst ecosystems, is important for both ecological theory and biogeochemical models. Specifically, the degree to which plant litter may be completely degraded whilst suspended on lichen surfaces and thus not enter the soil, which is a typical assumption of all biogeochemical models. We call for a shift in focus to cryptogam-mediated processes, rapid assessment of our findings more widely, and development of model parameters to complement the growing microbial and enzymatic focus of new biogeochemical models. not-yet-known not-yet-known not-yet-known unknown References Beckett, R.P., Minibayeva, F.V., Vylegzhanina, N.N., Tolpysheva, T., 2003. High rates of extracellular superoxide production by lichens in the suborder Peltigerineae correlate with indices of high metabolic activity. 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Soil enzymes as indicators of soil function: A step toward greater realism in microbial ecological modeling. Glob. CHANGE Biol. 28, 1935–1950. https://doi.org/10.1111/gcb.16036 Figures and tables. Figure 1. Typical context scenes of Cladonia arbuscula interaction with dwarf shrubs at the Geal charn research site. Left panels show Cladonia with Empetrum nigrum (top left), Arctostaphylos urva-ursi (centre left), Calluna vulgaris (bottom left). Example of litter physically connected to Cladonia aruscula thalli, with Calluna vulgaris and Arctostaphylos urva-ursi senesced leaf litter (top right), and magnified images of physical connection between Cladonia arbuscular thallus tips with senesced Calluna leaf (30 x magnification, centre right), and Calluna senesced stem-wood (35 x magnification, bottom right). not-yet-known not-yet-known not-yet-known unknown Figure 2. Activities of acid phosphatase (PHO), n-acetylglucosaminidase (NAG), xylosidase (XYL), and β-glucosidase (BG) in Cladonia arbuscula thalli from patches of Cladonia alone, and in association with shrubs (n=12 per situation). Square shows mean value, line shows median, box is 25th and 75th percentile, whiskers are 5th and 95th percentile. Table 1. P-values from Tukey’s post-hoc test of pairwise comparisons amongst contexts, where Cal = Calluna vulgaris ; Arcto = Arctostaphylos urva-ursi; Clad = Cladonia arbuscula; Emp = Empetrum nigrum. Bold text highlights differences p<0.05, enzyme abbreviations as described in text. Information & Authors Information Version history V1 Version 1 26 March 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords alpine decomposition extracellular glucosidase hydrolase lichen Authors Affiliations Robert Mills 0000-0002-4020-5129 [email protected] University of York View all articles by this author Josh Thurston University of York View all articles by this author Jake Spong 0000-0002-0815-0316 University of York View all articles by this author Metrics & Citations Metrics Article Usage 228 views 108 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Robert Mills, Josh Thurston, Jake Spong. Cladonia arbuscular holobiont systems produce enzymes that degrade plant and microbial biopolymers in an alpine shrub-heath.. Authorea . 26 March 2025. DOI: https://doi.org/10.22541/au.174298689.98349959/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. 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