Peer Review #1 of "Effects of auxin derivatives on phenotypic plasticity and stress tolerance in five species of the green alga Desmodesmus (Chlorophyceae, Chlorophyta) (v0.1)"
Green microalge of the genus Desmodesmus are characterized by a high degree of phenotypic plasticity, allowing them to be truly cosmopolitan and withstand environmental fluctuations. This flexibility enables Desmodesmus to produce a phenotype-environment match across a range of environments broader compared to algae with more fixed phenotypes. Indoles and their derivatives are a well-known crucial class of heterocyclic compounds and are widespread in different species of plants, animals, and
... roorganisms. Indole-3-acetic acid (IAA) is the most common, naturally occurring, plant hormone of the auxin class. IAA may behave as a signaling molecule in microorganisms, and the physiological cues of IAA may also trigger phenotypic plasticity responses in Desmodesmus. In this study, we demonstrated that the changes in colonial morphs of five species of the green alga Desmodesmus were specific to IAA but not to the chemically more stable synthetic auxins, naphthalene-1-acetic acid and 2,4-dichlorophenoxyacetic acid. Moreover, inhibitors of auxin biosynthesis and polar auxin transport inhibited cell division. Notably, different algal species (even different intraspecific strains) exhibited phenotypic plasticity different to that correlated to IAA. Thus, the plasticity involving individual-level heterogeneity in morphological characteristics may be crucial for microalgae to adapt to changing or novel conditions, and IAA treatment potentially increases the tolerance of Desmodesmus algae to several stress conditions. In summary, our results provide circumstantial evidence for the hypothesized role of IAA as a diffusible signal in the communication between the microalga and microorganisms. This information is crucial for elucidation of the role of plant hormones in planktonecology. Manuscript to be reviewed 12 Abstract 13 Green microalgae of the genus Desmodesmus are characterized by a high degree of phenotypic 14 plasticity (i.e. colony morphology), allowing them to be truly cosmopolitan and withstand 15 environmental fluctuations. This flexibility enables Desmodesmus to produce a phenotype-16 environment match across a range of environments broader compared to algae with more fixed 17 phenotypes. Indoles and their derivatives are a well-known crucial class of heterocyclic 18 compounds and are widespread in different species of plants, animals, and microorganisms. 19 Indole-3-acetic acid (IAA) is the most common, naturally occurring, plant hormone of the auxin 20 class. IAA may behave as a signaling molecule in microorganisms, and the physiological cues of 21 IAA may also trigger phenotypic plasticity responses in Desmodesmus. In this study, we 22 demonstrated that the changes in colonial morphs (cells per coenobium) of five species of the 23 green alga Desmodesmus were specific to IAA but not to the chemically more stable synthetic 24 auxins, naphthalene-1-acetic acid and 2,4-dichlorophenoxyacetic acid. Moreover, inhibitors of 25 auxin biosynthesis and polar auxin transport inhibited cell division. Notably, different algal 26 species (even different intraspecific strains) exhibited phenotypic plasticity different to that 27 correlated to IAA. Thus, the plasticity involving individual-level heterogeneity in morphological 28 characteristics may be crucial for microalgae to adapt to changing or novel conditions, and IAA 29 treatment potentially increases the tolerance of Desmodesmus algae to several stress conditions. 30 In summary, our results provide circumstantial evidence for the hypothesized role of IAA as a 31 diffusible signal in the communication between the microalga and microorganisms. This 32 information is crucial for elucidation of the role of plant hormones in plankton ecology. 33 34 Keywords Coenobial algae · Desmodesmus · Indole derivatives · Microalgae · Phenotypic 35 plasticity PeerJ reviewing PDF | Manuscript to be reviewed 37 Introduction 38 Phenotypic plasticity can be broadly defined as the capacity of a single genotype to exhibit 39 variable phenotypes in different environments and implies that a species can conquer diverse 40 environments. Phenotypic plasticity refers to some of the changes in an organism's behavior, 41 morphology and physiology in response to a unique environment. A well-known example of 42 phenotypic plasticity is changes in multicelled structures in coenobial algae. In these algae, 43 colonies reproduce asexually by successive divisions of the protoplast within the parent cell wall, 44 and when progeny are released, the parent wall remains. Daughter colony may be 45 morphologically identical to the parent, or they may exhibit remarkable phenotypic plasticity. 46 Most studies on phenotypic plasticity in coenobial algae have been conducted considering 47 morphological responses to an abiotic factor. Neustupa and Hodač (2005) demonstrated that 48 morphological plasticity of Pediastrum duplex var. duplex is related to the pH dynamics of 49 freshwater lakes. Peña-Castro et al. (2004) also reported the phenotypic plasticity in 50 Scenedesmus incrassatulus in response to heavy metal stress. However, microalgae are typically 51 associated with other microorganisms, such as zooplankton, fungi, and bacteria. Thus, studies on 52 phenotypic plasticity of the coenobial algae have increased in number and broadened their scope 53 from the focus on abiotic factors to biotic ones. Hessen and Van Donk (1993) first indicated that 54 the presence of the grazing pressure from water flea (Daphnia magna) can induce colony 55 formation in Scenedesmus algae. Furthermore, Lurling and his colleague proved that the induced 56 colony formation in the presence of herbivores is considered a strategy more efficient than 57 constitutive defenses under variable grazing risk (Lürling & Van Donk, 1996; Lürling, 2003). 58 Wu et al. (2013) further revealed that the number of cells per coenobium of Scenedesmus 59 increased with the population density of Daphnia growth, thus indicating a grazer density-60 dependent response. 61 Auxins, which constitute a class of plant hormones, have previously been suggested to 62 regulate physiological responses and gene expression in microorganisms (Spaepen et al., 2007). 63 Indole-3-acetic acid (IAA) is one of the most physiologically active auxins that can be produced 64 by numerous microbial species (Spaepen et al., 2007; Fu et al., 2015). Furthermore, phylogenetic 65 analyses have revealed that IAA biosynthetic pathways evolved independently in plants, bacteria, 66 algae, and fungi (Fu et al., 2015). The convergent evolution of IAA production leads to the 67 hypothesis that natural selection might have favored IAA as a widespread physiological code in 68 these microorganisms and their interactions. In natural water bodies, the crucial physical 69 associations and biochemical interactions between microalgae and other microorganisms are 70 generally well recognized (Natrah et al., 2014). Piotrowska-Niczyporuk and Bajguz (2014) found 71 that IAA plays a crucial role in the growth and metabolism of Chlorella vulgaris during a 72-72 hour culture period. Jusoh et al. (2015) indicated that IAA can induce changes in oil content, PeerJ reviewing PDF | Manuscript to be reviewed 73 fatty acid profiles, and expression of four genes responsible for fatty acid biosynthesis in Ch. 74 vulgaris at early stationary growth phase. In addition, the significance of these interactions in 75 algal phenotypic plasticity has attracted considerable scientific attention (Lürling & Van Donk, 76 1996; Lürling & Van Donk, 2000; Lürling 2003). Furthermore, IAA has been detected in some 77 species of Scenedesmaceae microalgae (Mazur et al., 2001; Prieto et al., 2011). We previously 78 used IAA as a signal molecule in microorganisms to simulate a selection pressure caused by 79 interspecific competition. The results indicated that the mean number of cells per particle of 80 Desmodesmus opoliensis and D. komarekii decreased gradually as the IAA concentration 81 increased gradually. The proportion of Desmodesmus unicells in monocultures increased with 82 IAA concentration. We also demonstrated that these unicells exhibited a lower tendency to 83 sedimentation than did large cells and that shrinkage may facilitate nutrient uptake and light 84 capture (Chung et al., 2018). However, whether other coenobial algal species of Desmodesmus 85 use the same strategy to overcome stress remains unknown. Hence, the objective of the present 86 study was to compare the effects of IAA at different concentrations on phenotypic responses in 87 different Desmodesmus species. Moreover, to address the auxin specificity of these processes 88 and obtain an insight into the complex auxin-related regulatory mechanism(s) in algal physiology, 89 we have selected a group of compounds called "auxin analogs," such as synthetically produced 90 naphthalene-1-acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), which are 91 structurally related to IAA. We thus aim to determine the differential effects of auxins and auxin-92 like compounds on the morphological responses of these coenobial algae. In addition, we 93 investigated the effects of inhibitors of auxin biosynthesis and auxin transport in Desmodesmus. 94 Here, 4-biphenylboronic acid (BBo), a potent YUCCA enzyme inhibitor and an Arabidopsis 95 growth inhibitor, and 2,3,5-triiodobenzoic acid (TIBA), a polar auxin transport inhibitor, were 96 used (Dhonukshe et al. 2008; Kakei et al. 2015). To elucidate the physiological changes induced 97 by phytohormone treatment, we also investigated whether IAA pretreatment promotes an 98 enhanced stress-tolerant phenotype. The obtained results are crucial for elucidating the role of 99 plant hormones in microalgal physiology. 100 101 Materials and Methods 102 Isolation and Culture of Microalgae 103 The algal strains used here were isolated from natural water bodies in Central Taiwan. Water 104 samples with visible microalgal population were centrifuged at 3000 ×g for 10 minutes at room 105 temperature to concentrate the cells and spread onto CA agar plates (with 0.8% w/v agar) (for 106 more details see Supplementary Materials). For isolating an axenic single colony from field 107 water samples, the streak plate method was used. The algae were cultured in CA medium. 108 Isolated algal cells were stored at −80°C in 15%-20% glycerol. For each experiment, the alga PeerJ reviewing PDF |