Light and Temperature Shape the Phenylpropanoid Profile of Azolla filiculoides Fronds

Abstract

Azolla is a genus of floating freshwater ferns. By their high growth and N2 fixation rates, Azolla species have been exploited for centuries by populations of South-east Asia as biofertilizers in rice paddies. The use of Azolla species as a sustainable plant material for diverse applications, such as feeding, biofuel production, and bioremediation, has encountered a growing interest over the last few years. However, high levels of feed deterrent flavonoids in their fronds have discouraged the use of these ferns as a sustainable protein source for animal consumption. Additionally, information on how and to what extent environmental determinants affect the accumulation of secondary metabolites in these organisms remains poorly understood. Moving from these considerations, here, we investigated by an untargeted metabolomics approach the profiles of phenylpropanoid compounds in the fronds of Azolla filiculoides sampled under control and pigment-inducing stress conditions. In parallel, we assayed the expression of essential structural genes of the phenylpropanoid pathway by quantitative RT-PCR. This study provides novel information concerning A. filiculoides phenylpropanoid compounds and their temporal profiling in response to environmental stimuli. In particular, we show that besides the already known 3-deoxyanthocyanidins, anthocyanidins, and proanthocyanidins, this fern can accumulate additional secondary metabolites of outstanding importance, such as chemoattractants, defense compounds, and reactive oxygen species (ROS) scavengers, and crucial as dietary components for humans, such as dihydrochalcones, stilbenes, isoflavones, and phlobaphenes. The findings of this study open an opportunity for future research studies to unveil the interplay between genetic and environmental determinants underlying the elicitation of the secondary metabolites in ferns and exploit these organisms as sustainable sources of beneficial metabolites for human health.

Advances and Future Prospects on Biotechnological Approaches Towards Azolla for Environmental Sustainability

“…Therefore, it has greater potential to achieve future sustainability goals defined by the United Nations through biotechnological approaches in a few main sectors, including animal feed, phytoremediation, and genomes (Figure 1). Azolla's biotechnology approaches have been well established in a variety of studies, such as phytoremediation (Ghorbanzadeh Mashkani, 2009;Goala et al, 2021;Talebi et al, 2019), enhancement of nutritive value (Brouwer et al, 2018;Costarelli et al 2021), phylogenetic analysis (Metzgar, 2007;Reid et al, 2006), and cryopreservation (Brouwer et al, 2014).…”

Exposure to different light intensities affects emission of volatiles and accumulations of both pigments and phenolics in Azolla filiculoides

“…Indeed, the synthesis of all phenolic compounds originating from the phenylpropanoid pathway, including anthocyanins, deoxyanthocyanidins (apigeninidin and luteolinidin), phlobaphenes, and soluble proanthocyanidins was particularly stimulated when A. filiculoides grown under low light was exposed to a 10‐fold higher light (Table 5 ; Figure S7C–J ). These phenolic compounds are responsible for the reddish leaf color observed in Azolla spp., especially in wintertime (Costarelli et al, 2021 ; Pieterse et al, 1977 ), being the red pigment luteolinidin 5‐ O glucoside recognized as a peculiar taxonomic marker of the genus Azolla (Iwashina et al, 2010 ). Leaf reddening is also common across higher plant species where it occurs as an adaptive mechanism to tolerate multiple environmental stresses (Hughes, 2011 ).…”

“…Plants of Azolla were bought from a local nursery in Pistoia (Italy) and further identified as Azolla filiculoides (Lam.) through molecular markers (Costarelli et al, 2021 ). The plants were grown in batch cultures by providing a liquid nutrient solution (as defined in Watanabe & Liu, 1992 ), replaced once a week to avoid nutrient limitation.…”

“…In particular, these ferns display leaf reddening upon the occurrence of stress (i.e. high light, low temperature) due to the accumulation of polyphenolic compounds (Costarelli et al, 2021 ; Kösesakal, 2014 ; Pieterse et al, 1977 ). Anthocyanins and other classes of flavonoids have been recognized to play multiple roles in photoprotection: they alleviate the photo‐oxidative damage to the chloroplasts by absorbing the excess of incident light and contribute to scavenging of oxidants and free radicals (Agati et al, 2020 ; Landi et al, 2015 ), thus maintaining reactive oxygen species (ROS) to the level required to exert a signaling function (Mittler, 2002 ).…”

High-resolution metabolite imaging: luteolinidin accumulates at the host-cyanobiont interface during cold-acclimation inAzollasymbioses

“…The main functions attributed to stress-induced red flavonoid pigment accumulation in land plants are photoprotection by filtering blue/green light, and ROS-scavenging (Davies et al, 2022). Consistently, excess light triggered DA-accumulation in A. filiculoides (Brilli et al, 2022), often in combination with abiotic stresses such as nutrient deficiency or cold (Tran et al, 2020; Costarelli et al, 2021). Prolonged cold-exposure may distort the carbon and nitrogen cycling within the symbioses and cause photooxidative damage that may result in starvation of the host.…”