Transcriptome profiling using RNA-Seq enabled comparing transcriptional response of two lettuce genotypes, Salinas and UC, grown in two contrasting nitrogen regimes. The results demonstrate the common and unique differential response of these genotypes. Although some stress responsive pathways were similar in both genotypes there were significant differences in the cultivated (Salinas) and the wild (UC) lettuce reaction to the abiotic stress conditions.
We found that nitrogen stress adversely affected several photosynthesis related genes such as those encoding for the light harvesting complex (LHC), the photosystem-I (PS-I) reaction center and RuBisCO. The LHC is associated with photosystem I and II which is made up of chlorophyll-a, chlorophyll-b, and binding proteins that act as light receptor to capture and deliver the excitation energy between PS-I and PS-II and also regulate the distribution of this energy under changing light conditions (Sage et al., 1988). Similar results were reported in bread wheat (Sultana et al., 2020) and in rice (Dalal and Tripathy, 2018) where nitrogen stress significantly decreased expression of components of LHCs of both PSII and PSI. Earlier studies have documented the effect of low nitrogen on physiological processes including low photosynthetic CO2 assimilation and decreased photosynthetic rate (Terashima and Evans, 1988; Rascher et al., 2000; Carmo-Silva et al., 2015), and also reduced nitrogen (Lawlor et al., 1987) and phosphorus (Simko, 2020) content in leaves. In addition, insufficient nitrogen can affect photosynthetic pigments (Xiaochuang et al., 2017), activity of Rubisco proteins (Kumagai et al., 2014; Carmo-Silva et al., 2015), and chlorophyll fluorescence (Carmo-Silva et al., 2015).
The photosynthetic response of the plant under abiotic stress can be quantified using chlorophyll fluorescence measurements. Our results showed that the photosynthetic efficiency (QY_max) and photosynthetic pigment concentration (SPAD values) were significantly lower under nitrogen stress conditions due to downregulation of genes involved in chlorophyll biosynthesis. Concomitantly, we observed a significant increase in non-photochemical quenching (NPQ) under nitrogen stress (Fig. 1). Thermal dissipation measured as NPQ, is a photoprotective mechanism that can eliminate excess irradiated energy absorbed by the plant. When plants absorb more light energy than they can utilize during abiotic stress, excess energy leads to the production of reactive oxygen species (ROS) which can cause severe damage to the plant’s photosynthetic apparatus and at higher levels can cause cell death. NPQ has been shown to increase under stresses conditions and plays an important role in the reduction in electron transport, increases in heat dissipation, and resistance to abiotic stresses (Zhao et al., 2016). Increases in NPQ in Salinas and UC genotypes suggest that dissipating the excess of excitation energy absorbed by PSII as heat is the principal pathway to reduce ROS formation under nitrogen stress conditions.
Nitrogen uptake, assimilation and metabolism and its effect on growth and biomass production have been studied in several agriculturally important plants (Masclaux-Daubresse et al., 2010; McAllister et al., 2012). The process of NO3¯ uptake, translocation and storage in plant is a complex process and is often controlled by low-affinity transporter (LAT) and high-affinity transporter (HAT) genes (Fan et al., 2017). Most nitrate transporter 1 genes act as nitrate sensors and function as low-affinity transporter genes for NO3− at high concentrations, with the exception of AtNRT1.1 in Arabidopsis and MtNRT1.1 in Medicago truncatula that serve as dual-affinity transporter involved in LAT and HAT systems (Liu and Tsay, 2003). Previous studies demonstrated upregulation of NRT1.1 upon addition of nitrate to nitrogen starved plants. For example, cultivar specific differences were observed in Brassica juncea for the expression of NRT1.1 and NRT1.8 that were highly induced as early as 20 min after exogenous supply of nitrate (Goel et al., 2018). In this study we did not find significant differences for Lettuce NRT1.1 gene expression under HN or LN condition confirming the role of NRT1.1 gene in nitrate perception. In contrast to our finding, Sultana et al. (2020) found that in wheat the expression of most of the dual affinity nitrate transporters (like NRT1.1) decreased under nitrogen stress and suggested that the reduced expression of NRT1.1 genes may lead to retarded growth, low grain protein content and low grain yield of nitrogen stressed plants. We observed that the expression of many high affinity nitrate transporter NRT2 genes was altered under low nitrogen conditions. The NRT2 genes family belong to HAT system and are responsible for transporting nitrate at low concentrations, therefore they play an important role during nitrogen stress conditions (Lezhneva et al., 2014; Fan et al., 2017; Zhang et al., 2018). Many NRT2 family members require NAR2 (nitrate assimilation related protein) for nitrate transportation and have different spatio-temporal distribution in roots (Kiba et al., 2012; Lezhneva et al., 2014; Kiba and Krapp, 2016). In Arabidopsis, NRT2.1 mediates apoplastic nitrate absorption and affects root system architecture under low nitrogen conditions (Remans et al., 2006; Zhang et al., 2018). Unlike NRT2.1, NRT2.4 and NRT2.5 are expressed in epidermal cells of roots under long term nitrogen starvation and responsible for nitrate uptake from soil into the plant (Kiba et al., 2012; Lezhneva et al., 2014). We found elevated expression of several high affinity nitrate transporter lettuce genes (NRT2.1, NRT2.4 and NRT2.5) in UC genotype under low nitrogen condition which may explain higher nitrogen content observed in UC than Salinas genotype thus indicating that UC genotype could have higher nitrogen uptake efficiency under low nitrogen conditions.
The process of converting inorganic nitrogen from soil into organic nitrogen is carried out by two key enzymes, glutamine synthase (GS) and glutamate synthase (GOGAT). GS catalyzes adenosine triphosphate (ATP)-dependent fixation of ammonium to form glutamine while the GOGAT enzyme catalyzes the conversion of glutamine to two molecules of glutamate (Temple et al., 1998). Glutamine serves as a main nitrogen donor for biosynthesis of amino acids, nucleotides, and chlorophyll. Therefore the GS enzyme is the major factor affecting nitrogen assimilation in plants (Cai et al., 2009). There are two isoenzyme forms of the GS enzyme, one located in the cystosol (GS1 or GLN1) and the other in chloroplast/plastids (GS2 or GLN2). The cytosolic GLN1 plays an important role in nitrogen assimilation in roots while the chloroplastic GLN2 is expressed primarily in leaves and is responsible for the reassimilation of the ammonia generated by photorespiration (Cai et al., 2009). In this study, we found that the expression of genes for GLN1 was up-regulated under nitrogen stress in both genotypes while the expression of GLN2 was higher in Salinas under high nitrogen but was down-regulated under nitrogen stress. There was no change in the expression levels of the GLN2 in UC under high or low nitrogen conditions. Together, these results suggest that the UC can sustain nitrogen assimilation under low nitrogen condition and may account for higher nitrogen content and biomass produced during nitrogen stress.
Transcription factors (TFs) recognize specific motifs and function as a switch that can turn on or off a particular gene. In this study we found that the expression of TFs like bHLH, bZIP, ERF, MYB, NAC, and WRKY was altered in response to nitrogen stress (Supplementary_Fig. 4). The Apetala2/ethylene responsive factors (AP2/ERF) family TFs are the key regulators of numerous abiotic stresses and respond to several plant hormones (Mizoi et al., 2012; Chandler, 2018). These TFs are induced upon specific stresses and have diverse DNA binding preferences, enabling these TFs to integrate responses of multiple stimuli and participate in regulatory processes (Xie et al., 2019). Here we found that of the 14 ERF transcription factor showing differential expression, 12 were upregulated in both, Salinas and UC under low nitrogen condition suggesting ERF TFs play a crucial role in regulating nitrogen responsive genes in lettuce. Similar results were observed in cucumber by Zhao et al. (2015b), where they identified over seven differentially expressed ERF TFs in response to early nitrogen deficiency. The basic leucine zipper (bZIP) TF family is one of the largest TF family in plants that is involved in regulating plant growth, development, and biotic and abiotic stress responses (Jakoby et al., 2002). In this study, the majority of the bZIP TFs were identified in the Salinas genotype with both, up- and down-regulation of their expression in response to nitrogen stress. Functional characterization of a bZIP TF (TabZIP) in wheat revealed it regulated several biological processes including cellular nitrogen compound metabolic process (GO:0034641) (Agarwal et al., 2019). In Arabidopsis, a bZIP TF AtTGA4 was found to be induced under drought and low nitrogen stress and its over-expression resulted in improved drought tolerance and reduced nitrogen starvation (Zhong et al., 2015). The transgenic plant overexpressing AtTGA4 had higher nitrogen and proline content than the wild type controls. The activity of nitrogen transportation (NRT2.1, NRT2.2) genes and nitrogen assimilation (NIA1, NIA2) genes were also higher in the transgenic plants. We observed that the expression of NIA2 gene (Ls8_106740.1) was significantly higher under nitrogen stress and may be under bZIP TF regulation. The number of downregulated basic helix-loop-helix domain (bHLH) TFs were greater in Salinas than the UC genotype. Plant bHLH proteins control response to light and interact with components of the circadian clock (Hanano et al., 2008). In Arabidopsis AtbHLH008 can regulate circadian clock genes CCA1 and LHY thus providing entry point to phytochrome regulation of the circadian clock (Martı́nez-Garcı́a et al., 2000). Downregulation of bHLH genes in Salinas under stress maybe a mechanism to minimize photoreception during reduced photosynthesis thereby protecting plant from ROS production. The NAC TFs control several biochemical and molecular pathways that help plants to survive under different stress conditions. In this study we found that the differentially expressing NAC TFs were upregulated in both genotypes and none were identified to be downregulated (Fig. S4). He et al. (2015) identified a NAC TF TaNAC2-52 in wheat that could bind to the promoter regions of the genes encoding nitrate transporter and glutamine synthase. Overexpression of TaNAC2-52 TF caused enhanced root growth with higher nitrate influx rate, resulting in higher nitrogen accumulation in aerial parts of the transgenic wheat plants. Similar results were reported for TaNAC-S TF where its over expression resulted in delayed leaf senescence (stay green phenotype) and higher nitrogen/protein content in grains of the transgenic wheat plants (Zhao et al., 2015a).
Genes with similar pattern of expression are most likely to be under similar regulation (Allocco et al., 2004). In this study, we used WGCNA to construct gene co-expression networks and to identify hub gene. All DEGs were divided into nine modules, each of them significantly correlated with one or more phenotypic traits. Co-expression network construction highlighted several hub genes that are expected to play important roles during nitrogen stress in lettuce. All the correlated networks identified in this study were regulated by one or more transcription factors. Transcriptional regulation of nitrogen use efficiency genes is reported in tea (Zhang et al., 2020), soybean (Hao et al., 2011), Arabidopsis (Zhuo et al., 1999), brassica (Goel et al., 2018), bean (Lardi et al., 2017). The carbon assimilation gene and high affinity nitrate transporter genes were identified in the lightgreen module cluster regulated by ERTFs (RAP2-11, ERF098) that are associated with the biological function of response to ROS and are involved in regulation of gene expression by stress factor and by components of stress signal transduction. Regulation of the nitrate transporter genes by RAP2-11 was demonstrated in Arabidopsis (Meng et al., 2016). Similar results were also observed in cucumber where the ERF TFs regulated photosynthesis related genes, nitrogen deficiency responsive genes and carbon assimilation genes (Zhao et al., 2015b).
A major hub gene Ls5_44120.1 encoding for PLAT domain-containing protein 2 (PLAT2) appear to be interacting with large number of genes in the blue module cluster. In Arabidopsis the PLAT 1 gene is shown to be regulated by bZIP TF and is critically involved in abiotic stress tolerance (Hyun et al., 2014). Two hub genes, Ls5_189641.1 and Ls2_60101.1 interacted with many genes in the second cluster of the blue module. The gene Ls5_189641.1 encoding for Glutaredoxin-C13 (GRXC13) is associated with cellular response to nitrogen starvation and has been shown to mediate signaling and plant response to nitrate starvation in Arabidopsis (Jung et al., 2018) while the gene Ls2_60101.1 for cationic amino acid transporter 5 protein (CAT5) is involved in transport of the amino acids. The CAT genes are part of amino acid transporters (AATs), are protein that perform inter- and intra-cellular movements of amino acids after nitrogen assimilation therefore are important for nitrogen homeostasis in plants (Su et al., 2004; Lu et al., 2012; Feng et al., 2018). Altered expression of these hub genes may account for the positive and negative correlations of the lightgreen and blue modules with the phenotypic traits including the photosynthetic efficiency of PSII, SPAD, and total nitrogen content in lettuce.