Genetic evidence for algal auxin production in Chlamydomonas and its role in algal-bacterial mutualism

Abstract

Interactions between algae and bacteria are ubiquitous and play fundamental roles in nutrient cycling and biomass production. Recent studies have shown that the plant auxin indole acetic acid (IAA) can mediate chemical crosstalk between algae and bacteria, resembling its role in plant-bacterial associations. Here, we report a mechanism for algal extracellular IAA production from L-tryptophan mediated by the enzyme L-amino acid oxidase (LAO1) in the model Chlamydomonas reinhardtii. High levels of IAA inhibit algal cell multiplication and chlorophyll degradation, and these inhibitory effects can be relieved by the presence of the plant-growth-promoting bacterium (PGPB) Methylobacterium aquaticum, whose growth is mutualistically enhanced by the presence of the alga. These findings reveal a complex interplay of microbial auxin production and degradation by algal-bacterial consortia and draws attention to potential ecophysiological roles of terrestrial microalgae and PGPB in association with land plants.

Keywords: Microbiology; Plant biochemistry; Plant biology.

Graphical abstract

Figure 1

L-amino oxidase (LAO1) plays a critical role in indole-3-acetic acid (IAA) production from L-tryptophan (L-Trp) in Chlamydomonas Chlamydomonas wild-type (dark green circles) and lao1 null mutant (light green triangles) log-phase cells at 5×10⁶ cells/ml were incubated for 48 h in nitrogen-free medium supplemented with 5 mM L-Trp as a sole source of nitrogen. This is the minimum concentration at which we observed a significant growth reduction in the absence of any other N source available (Figure S1C). (A and D) L-Trp, (B and E) indole-3-pyruvic acid (IPyA), and (C and F) IAA were quantified in the cell-free supernatants by HPLC; the identity of these compounds was confirmed with LC-MS/MS. The MS2 match was performed using the Fragment Ion Search function of Compound Discoveror 3.1 (Thermo Fisher Scientific, San Jose, USA). Green dots represent MS2 matchings for L-Trp (B), IPyA (D), and IAA (F). (G) In the periplasm, LAO1 and LAO2/RIDA (Reactive Intermediate/Imine Deaminase A) deaminate extracellular L-Trp to produce the α-keto acid IPyA, which is decarboxylated to IAA by means of a yet unidentified mechanism (dashed arrows).

Figure 2

IAA arrests cell multiplication and attenuates chlorophyll degradation in nitrogen-limited Chlamydomonas (A–C) Impact of exogenously added tryptophan (L-Trp), indole-3-pyruvic acid (IPyA), and indole-3-acetic acid (IAA) on Chlamydomonas growth in the presence of L-alanine (L-Ala). Wild-type cells at an initial concentration of 0.2×10⁶ cells/mL were grown for three days on L-Ala (4 mM) as an N source (to enable algal growth under N-limiting conditions and ensuring LAO1 expression; see STAR Methods) in the presence of the indicated concentrations of (A) L-Trp, (B) IPyA, or (C) IAA. Cell culture densities are indicated as solid lines and chlorophyll content per cell as dotted lines. (D–F) Impact of IAA in Chlamydomonas during N deprivation. (D) Cell density and (E) chlorophyll content during N deprivation (in the absence of any assimilable N source). Wild-type cells at 10⁶ cells/ml were incubated in N-free media (–N) or supplemented with 500 μM of IAA (IAA). Data are averages of 3 biological replicates with error bars depicting standard deviations. Asterisks indicate statistically significant differences compared with the control without IAA (t test: n = 3; ∗α < 0.05; ∗∗α < 0.005; ∗∗∗α < 0.001). (F) A representative culture flask of each condition in panels (D) and (E) was imaged at the start, after one day, and after five days; cell pellets shown were harvested by centrifuging 0.5 mL of the cultures after five days of incubation.

Figure 3

Methylobacterium aquaticum reduces IAA levels in Chlamydomonas cultures to relieving algal inhibition of cell multiplication and chlorophyll degradation (A) Algal cell density and (B) chlorophyll content per cell were determined initially (0 days) and after five days of incubation for Chlamydomonas monocultures (Cre) and Chlamydomonas-M. aquaticum co-cultures (Cre-Maqu) in N-free medium (–N) supplemented with 500 μM IAA (IAA). Initial cell concentrations were 10⁶ cells/ml for Chlamydomonas and A₆₀₀ of 0.01 for M. aquaticum (approximately 10⁶ cells/mL). Chlamydomonas monocultures correspond to the same dataset as in Figures 2D and 2E and are represented here as a reference for algal monocultures. (C) Chlamydomonas (Cre) monocultures, M. aquaticum (Maqu) monocultures, and co-cultures (Cre-Maqu) were incubated on N-free media supplemented with 500 μM of IAA for five days. Indoles concentration in the cell-free media were determined using the Salkowski reagent (see STAR Methods). (D) Algal and bacterial cell densities after eight days of growth on 500 μM of IAA in coculture or monoculture were quantified using qPCR of single-copy genes specific for Chlamydomonas (centrin) or M. aquaticum (rpoB) (see STAR Methods). Data shown are averages of 3 biological replicates with error bars depicting standard deviations. Asterisks indicate statistically significant differences compared with the control comparison (t test: n = 3; ∗α < 0.05; ∗∗α < 0.005; ∗∗∗α < 0.001). (E) Chlamydomonas-M. aquaticum cocultures were imaged using a light microscope under conditions without a nitrogen source (-N) and (F) supplemented with 500 μM IAA (IAA) after five days.

Figure 4

Proposed role of auxin-mediated mutualistic interactions between Chlamydomonas, Methylobacterium, and land plants For a Figure360 author presentation of this Figure 4, see https://doi.org/10.1016/j.isci.2023.108762. In soil environments where tryptophan may be present due to plant exudation and microbial decay, bacterial indole-3-acetic acid (IAA) biosynthesis from this amino acid can promote plant growth. Chlamydomonas reinhardtii can also convert tryptophan into IAA using the extracellular enzyme LAO1. Accumulation of this auxin may result in algal growth arrest and in attracting beneficial PGPB bacteria. In the presence of the IAA-degrading bacterium Methylobacterium aquaticum, IAA is depleted, enhancing growth of both microorganisms. Metabolites exchanged between bacteria and algae could strengthen this mutualistic interaction. Moreover, we imagine three-way algal-plant-bacterial associations whereby algal-derived IAA not only benefits the alga but promotes plant-bacterial symbioses and modulates plant physiology directly. Figure created using BioRender.com and ChemDraw 20.1. [∗] indicates interactions or processes shown in this work. Numbers in square brackets correspond to references: [1] Vallon et al.; [2] Cox et al.^,,; [3] Rico-Jiménez et al.; [4] Kravchenko et al.; [5] Moe LA; [6] Backer et al.