Duckweed roots are dispensable and are on a trajectory toward vestigiality

Duckweeds are morphologically simplified, free floating aquatic monocots comprising both rooted and rootless genera. This has led to the idea that roots in these species may be vestigial, but empirical evidence supporting this is lacking. Here we show that duckweed roots are no longer required for their ancestral role of nutrient uptake. Comparative analyses of nearly all rooted duckweed species revealed a highly reduced anatomy, with greater simplification in the more recently diverged genus Lemna. A series of root excision experiments demonstrated that roots are dispensable for normal growth in Spirodela polyrhiza and Lemna minor. Furthermore, ionomic analyses of fronds in these two species showed little difference in the elemental composition of plants in rooted versus root-excised samples. In comparison, another free-floating member of the Araceae, Pistia stratiotes, which colonized the aquatic environment independently of duckweeds, has retained a more complex root anatomy. Whilst Pistia roots were not absolutely required for growth, their removal inhibited plant growth and resulted in a broad change in the mineral profile of aerial tissues. Collectively, these observations suggest that duckweeds and Pistia may be different stages along a trajectory towards root vestigialisation Given this, along with the striking diversity of root phenotypes, culminating in total loss in the most derived species, we propose that duckweed roots are a powerful system with which to understand organ loss and vestigiality.

The Dynamics of NO3− and NH4+ Uptake in Duckweed Are Coordinated with the Expression of Major Nitrogen Assimilation Genes

Duckweed plants play important roles in aquatic ecosystems worldwide. They rapidly accumulate biomass and have potential uses in bioremediation of water polluted by fertilizer runoff or other chemicals. Here we studied the assimilation of two major sources of inorganic nitrogen, nitrate (NO3−) and ammonium (NH4+), in six duckweed species: Spirodela polyrhiza, Landoltia punctata, Lemna aequinoctialis, Lemna turionifera, Lemna minor, and Wolffia globosa. All six duckweed species preferred NH4+ over NO3− and started using NO3− only when NH4+ was depleted. Using the available genome sequence, we analyzed the molecular structure and expression of eight key nitrogen assimilation genes in S. polyrhiza. The expression of genes encoding nitrate reductase and nitrite reductase increased about 10-fold when NO3− was supplied and decreased when NH4+ was supplied. NO3− and NH4+ induced the glutamine synthetase (GS) genes GS1;2 and the GS2 by 2- to 5-fold, respectively, but repressed GS1;1 and GS1;3. NH4+ and NO3− upregulated the genes encoding ferredoxin- and NADH-dependent glutamate synthases (Fd-GOGAT and NADH-GOGAT). A survey of nitrogen assimilation gene promoters suggested complex regulation, with major roles for NRE-like and GAATC/GATTC cis-elements, TATA-based enhancers, GA/CTn repeats, and G-quadruplex structures. These results will inform efforts to improve bioremediation and nitrogen use efficiency.

Light intensity drives different growth strategies in two duckweed species: Lemna minor L. and Spirodela polyrhiza (L.) Schleiden

Duckweed species Lemna minor and Spirodela polyrhiza are clonal plants with vegetative organs reduced to a frond and a root in L. minor or a frond and several roots in S. polyrhiza. They reproduce vegetatively by relatively rapid multiplication of their fronds. The habit of S. polyrhiza (large fronds with up to 21 roots) makes it a strong competitor among representatives of the family Lemnaceae, probably due to different resource-use strategies compared to small duckweed. In our study, light was the resource that affected the plants before and during the laboratory experiment. We sampled the plants from natural habitats differing in light conditions (open and shady) and grew them for 16 days in a thermostatic growth room at 22 °C under a 16:8 photoperiod and three light intensities (125, 236, 459 µmol photons m–2 s–1) to investigate the trade-off between frond enlargement and multiplication. Both species from the open habitat had higher growth rates based on the frond numbers and on surface area of fronds compared to plants from the shady habitat. They adopted different species-specific strategies in response to the experimental light conditions. The species size affected the growth rates in L. minor and S. polyrhiza. Spirodela polyrhiza grew slower than L. minor, but both species grew fastest at medium light intensity (236 µmol m–2 s–1). Lemna minor maintained the growth rates at high light intensity, while S. polyrhiza slowed down. Spirodela polyrhiza responded to deteriorating light conditions by increasing its frond surface area, thus optimising light capture. Lemna minor from the shady habitat enhanced light harvest by increasing chlorophyll a concentration, but did not invest more in frond enlargement than L. minor from the open habitat. Under shady conditions, S. polyrhiza is likely to achieve an advantage over L. minor due to the larger frond size of the former. Our findings suggest the existence of a trade-off between size and number in duckweed.

Growth promotion of giant duckweed Spirodela polyrhiza (Lemnaceae) by Ensifer sp. SP4 through enhancement of nitrogen metabolism and photosynthesis

Duckweeds (Lemnaceae) are representative producers in fresh aquatic ecosystems and also yield sustainable biomass for animal feeds, human foods, and biofuels, and contribute toward effective wastewater treatment, thus enhancing duckweed productivity is a critical challenge. Plant growth-promoting bacteria (PGPB) can improve the productivity of terrestrial plants; however, duckweed–PGPB interactions remain unclear and no previous study has investigated the molecular mechanisms underlying duckweed–PGPB interaction. Herein, a PGPB, Ensifer sp. strain SP4, was newly isolated from giant duckweed (Spirodela polyrhiza (L.) Schleid.), and the interactions between S. polyrhiza and SP4 were investigated through physiological, biochemical, and metabolomic analyses. In S. polyrhiza and SP4 co-culture, SP4 increased the nitrogen (N), chlorophyll, and RuBisCO contents and the photosynthesis rate of S. polyrhiza by 2.5-, 2.5-, 2.7-, and 2.4-fold, respectively. Elevated photosynthesis increased the relative growth rate and biomass productivity of S. polyrhiza by 1.5- and 2.7-fold, respectively. SP4 significantly altered the metabolomic profile of S. polyrhiza, especially its amino acid profile. N stable isotope analysis revealed that organic N compounds were transferred from SP4 to S. polyrhiza. These N compounds, particularly glutamic acid, possibly triggered the increase in photosynthetic and growth activities. Accordingly, we propose a new model for the molecular mechanism underlying S. polyrhiza growth promotion by its associated bacteria Ensifer sp. SP4, which occurs through enhanced N compound metabolism and photosynthesis. Our findings show that Ensifer sp. SP4 is a promising PGPB for increasing biomass yield, wastewater purification activity, and CO2 capture of S. polyrhiza.