Metabolite profiling reveals insights into the species-dependent cold stress response of the green seaweed holobiont Ulva (Chlorophyta)

Abstract

The green seaweed Ulva (Chlorophyta) and its associated epibacterial microbiome form a functional holobiont that adapts to stress as a whole. As the macroalga provides carbon sources for bacteria and relies on algal growth and morphogenesis-promoting factors (AGMPF) released by bacteria, those cross-kingdom interactions are especially fascinating. We hypothesized that the Ulva holobionts from the warm-temperate Mediterranean-Atlantic and cold Antarctic habitats respond to cold stress differently depending on the production of highly polar low molecular weight compounds (LMWCs) with stress-regulating activity. We compared the microbiome of and metabolic changes in U. compressa (cultivar U. mutabilis ), initially collected in Ria Formosa (Portugal), with that of U. bulbosa (strain AWI #1002) collected in Antarctica by performing a warm-cold temperature shift experiment. Microbiome analysis indicated significant differences between the two species and that the number of operational taxonomic units (OTUs) was lower in cultivated U. bulbosa than in freshly isolated Ulva ; despite this, AGMPF-producing bacteria were detected in both holobionts. Significant differences in metabolite profiles were observed between both species using hydrophilic interaction liquid chromatography coupled with electrospray ionization mass spectrometry (HILIC-ESI-MS). Biomarkers such as dimethylsulfoniopropionate (DMSP) and taurine were identified following a temperature shift from 18 °C to 5 °C in the warm-temperate U. mutabilis and the cold-adapted U. bulbosa , respectively. Our findings show that metabolic changes in the holobiont in response to cold are species-dependent. To evaluate the contribution of the metabolic changes of bacteria and algae to the stress response, the reductionistic model system of the tripartite community formed by U. mutabilis and its two essential bacteria, Roseovarius sp. strain MS2 and Maribacter sp. strain MS6, which release all essential AGMPFs, was investigated. We examined the production of polar LMWCs in the presence and absence of bacteria following a shift to cold temperatures. Among the metabolites studied, ectoine ((4 S )-2-methyl-3,4,5,6-tetrahydropyrimidine-4-carboxylic acid) was only detected in the presence of bacteria, highlighting the role of bacteria in releasing compounds that mitigate environmental stresses through cold stress adaptation factors (CSAF). Our findings suggest that microbiome engineering will allow us to improve macroalgae adaptability to stressful situations, which can be further applied to the sustainable management of (land-based) aquaculture systems. • Algal growth and morphogenesis promoting bacteria were found in cultures of Ulva independently of their origin and habitat. • Low molecular weight compounds (LMWC) of the holobiont were examined upon cold stress. • Cold stress triggers diverse potential cryoprotectants in warm- and cold-adapted Ulva. • Taurine and DMSP may be considered as species-specific stress biomarkers. • Axenic Ulva cultures elucidate the bacterial contribution to the production of LMWC.