Temporal variations in the expression of a diatom nitrate transporter gene in coastal waters off northern Taiwan: The roles of nitrate and bacteria

Highlights

• This study investigated the expression level of a nitrate transporter gene, Nrt2, in a marine diatom.

• RNA-Seq was used to identify Nrt2 sequences in a dominant diatom, and the expression of Nrt2 was measured by qPCR.

• Local [NO₂⁻ + NO₃⁻] displayed a clear seasonal cycle and influenced Nrt2 expression in the diatom examined.

• Bacteria and nanoflagellates correlated with diatom Nrt2 expression, suggesting competition and regeneration effects.

Abstract

In this study, the mRNA levels of a diatom nitrate transporter gene, Nrt2, were investigated in a coastal ecosystem on the western boundary of the northwestern Pacific Ocean. During the period between June 2011 and June 2013, 14 cruises were conducted, and samples were collected for nutrient-manipulation incubations, quantitative polymerase chain reactions (qPCRs), and metatranscriptome construction. Based on metatranscriptomes generated through next-generation sequencing, the dominant diatoms and sequences associated with Nrt2 as well as EFL, a qPCR reference gene, were identified. Next, primer pairs for a dominant bloom-forming diatom, Chaetoceros cf. curvisetus, were designed, and the expression levels of Nrt2 were measured by qPCR in both original (untreated) and nitrogen-deprived samples. By calculating the ratio of Nrt2 expression between these 2 samples, a Log (f_Nrt2) index was developed to be used as a molecular indicator of nitrogen demand. At our study site, the nitrate and nitrite concentrations displayed a clear seasonal cycle, with high (8.75 μM) and low (0.05 μM) concentrations occurring in December and June, respectively. The Log (f_Nrt2) index also varied with time in 2012, with a high value of approximately −1.44 occurring in October or November; this variation was not in phase with the nitrate trend. The regression analysis indicated that Log (f_Nrt2) was inversely correlated with nitrate and nitrite concentrations. On the other hand, Log (f_Nrt2) was positively correlated with bacterial abundance. This regression equation properly included values of Log (f_Nrt2) that were not well explained by the nitrate and nitrite concentrations. When the Chaetoceros abundance increased, the associated low value of Log (f_Nrt2) suggested the repression of Nrt2 by ammonium; apparently, the diatom utilizes ammonium as the nitrogen source to maintain its dominance in the later phase of the bloom. Since bacteria and nanoflagellates are actively involved in the uptake and release of nitrogenous nutrients, these two functional groups also control the expression of Nrt2 through important ecological processes such as competition and regeneration.

Introduction

Marine diatoms account for more than 20% of global CO₂ fixation and nearly 40% of marine primary production (Roberts et al., 2007). Due to their large size, diatoms are readily consumed by crustacean zooplankton. Thus, the abundance of diatoms is important for energy transfer throughout the grazing food chain (Falkowski et al., 2004). In addition, some ingested diatoms are packed into fecal pallets. The sinking carbon flux that occurs through this pathway contributes significantly to the global carbon cycle (Karl et al., 2012).

The growth rate and biomass of diatoms in marine environments are often limited by nitrogen (N), iron (Fe), or phosphorus (P). Among them, N and Fe limitations occur more frequently (Downing et al., 1999). In the utilization of nitrogenous nutrients, ammonium is the chemical form preferred by microalgae (Levasseur et al., 1993; Roopnarain et al., 2015). However, since the ammonium concentration in seawater is usually low, nitrate is still the major form that supports phytoplankton growth (Kanda et al., 1990). According to studies in higher plants, the uptake of nitrate depends on a group of nitrate transporter proteins (NRTs) that control the first step in the process of nitrogen utilization. NRT proteins include a low-affinity transporter system (LATS) and a high-affinity transporter system (HATS). The LATS consists of proteins in the NRT1/PTR family (recently renamed the NPF family), and LATS proteins are activated when the nitrate concentration is greater than 1 mM (Crawford and Glass, 1998; Galvan and Fernandez, 2001; Glass et al., 2002; Okamoto et al., 2003). On the other hand, The HATS contains the NRT2/NNP protein family (Sun and Zheng, 2015). Kinetic studies indicate that HATS proteins can be further classified into a nitrate-inducible transport system (iHATS) and a constitutively expressed system (cHATS). In Arabidopsis thaliana, cHATS and iHATS typically operate within an NO₃⁻ concentration range of 10–250 μM.

In early studies among phytoplankton, genes encoding LATS proteins were mainly reported in the genomes of green algae: Chlamydomonas reinhardtii, Ostreococcus tauri, and Micromonas spp. (Fernandez and Galvan, 2007; McDonald et al., 2010). Recently, LATS-related genes belonging to the “nitrate transporter 1/peptide transporter family (NPF)” have been reported in several species of diatoms (known as diNPFs) (Rogato et al., 2015; Santin et al., 2021). However, NPFs have been shown to recognize a broad range of substrates, which included nitrate and peptides in higher plants (Léran et al., 2014). Whether diNPFs function as a typical LATS requires further investigations of their enzymatic activity, substrate specificity, and roles in diatom nitrogen metabolism. On the other hand, genes encoding HATS proteins (NRT2 family) have also been reported in diatoms (Hildebrand and Dahlin, 2000; Kang and Rynearson, 2019). The functions of diatom HATS are better characterized via the experimental techniques of ¹⁵N uptake and nitrate induced gene expression (Smith et al., 2019).

In a transcriptomic analysis, gene expressions of 3 species of model diatoms were compared under the treatment of nitrate limitation (Bender et al., 2014). The expression levels of genes related to nitrogen utilization were affected, but a common trend was absent among species. When genes in the NRT2 family were considered separately, Bender et al. (2014) discovered that individual diatom species often contained multiple forms of Nrt2, and that, in most cases, the treatment of nitrate limitation increased the expression of these genes. An analysis of the Tara Oceans eukaryotic metatranscriptomes indicated that the mRNA abundances of certain diatom Nrt2 clades were commonly high on a global scale, but transcript abundances of the ammonium transporter gene, AMT1, were divergent across geographic regions (Busseni et al., 2019). This observation suggests that individual diatom species at different locations may contain Nrt2 genes with similar expression patterns and biological functions.

In experimentally examined diatom species, the expression levels of nitrate transporter genes are closely related to the ambient nitrogen concentrations (Kang et al., 2009). The expression of Nrt2, a nitrate transporter gene, increased slightly under nitrate-sufficient conditions and rose substantially to a much higher level under nitrogen starvation conditions. The regulation of Nrt2 is also affected by the concentration of environmental ammonium. When diatom cells take up ammonium as the nitrogen source, Nrt2 expression is severely repressed (Kang et al., 2009). Sometimes, the repressed mRNA level may become lower than the detection limit of RT-qPCR. Some other factors, such as light, temperature (Bender et al., 2012), and phytoplankton-grazer interactions (Amato et al., 2018) may also affect the regulation of NO₃⁻ and NH₄⁺ uptake. However, nutrient manipulation experiments conducted in the East China Sea showed that diatom abundance and Nrt2 expression were closely related to nutricline depth (Kang et al., 2015). Therefore, Nrt2 expression is regarded as a useful indicator of the status of nitrogen utilization in natural populations of diatoms (Suzuki et al., 2019).

In this study, we investigated a coastal ecosystem on the western border of the northwestern Pacific Ocean to reveal the temporal variation in Nrt2 expression in a diatom, Chaetoceros cf. curvisetus. Using samples collected by a plankton net, metatranscriptomes were constructed, and sequence analysis was employed to identify dominant diatoms and Nrt2-related genes. Subsequently, the expression patterns of Nrt2 were measured by RT-qPCR with carefully designed primer pairs. By examining factors that correlated with Nrt2 expression, ecological processes leading to nitrogen stress in this diatom were discussed.