Activation of MAPK signaling pathway during nitrogen-deficiency responses in Ulva Prolifera CURRENT STATUS:

Background: Ulva prolifera is one of the main seaweeds (or macroalgae) species that causes “green tides”. This alga inhabits the estuarine areas that exhibit changes in nutrient contents, which include changes in nitrogen (N) levels, while the mechanisms through which these microalgae resist N deficiency remains unclear. Results: We amplified the full-length sequences and quantified expression of genes involved in the nitrogen metabolism process, the data indicated that nitrate reductase, nitrite reductase, and glutamine synthase increased after nitrogen deprivation in Ulva prolifera. Hence, although the ratio of cell-wall regeneration did not change, the apoptosis rates of protoplasts of Ulva prolifera increased after this deficiency. Furthermore, a decreased in N supplies triggered the activation of MAPK signaling, and SB239063, a p38 MAPKα/β inhibitor, enhanced the effects of N deficiency on the mortality of protoplasts and decreased the capacity for cell-wall regeneration. Conclusions: All the data provided evidence that MAPK signaling had functional roles in helping U. prolifera adapt to fluctuations in N availability within a short time. Hence, the application of biochemical reagents on cell-wall regeneration on the surface of protoplasts provided a new perspective in the genetic breeding of Ulva prolifera.

nitrate concentrations [11], but the mechanisms by which nitrate assimilates in the species that form green tides has been unclear.
Among the major sources of nitrogen (N) for plant growth and development, nitrate plays a more important role than ammonium. In plants, nitrate reductase (NR) and nitrite reductase (NiR) catalyze nitrate into nitrite, glutamine synthetase (GS), and glutamine oxoglutarate aminotransferase (GOGAT) and are involved in ammonium assimilation [12]. The function of these enzymes contributes to eliminating the toxic effects of nitrite and ammonium produced under biotic and abiotic stress. For example, 50 min of N depletion enhances the levels of NR activity in Ankistrodesmus braunii [13]; NR mRNA accumulation fluctuates during a light-dark cycle in both tomato and tobacco plants, which rapidly increases during dark periods and reaches a maximum at the beginning of the light period [14]. NR establishes the connections between nitrate assimilation and photosynthesis in a number of unicellular marine algae under N-limiting conditions, which promotes the uptake of inorganic and organic N [15]. Although these enzymes have been widely identified in higher plants, because of poor genome and molecular studies on algae, this is the first time that the functional effects of UpNR, UpNiR, and UpGOGAT have been demonstrated to prevent N deficiency from having toxic effects on U. prolifera.
Hence, except for the genes mentioned above, some nutrient signaling has also been intensively studied during recent years [16,17]. For example, an increase in the steady-state level of mRNA was observed for MPK11 and MKK9 in response to N deficiency [18]; N deficiency was observed to induce Ras 1 phosphorylation at Ser-226 residues, which regulate the growth and proliferation of budding yeast Saccharomyces cerevisiae [19]; the dephosphorylation of Sch9, a direct substrate of the yeast multiprotein complex target of rapamycin (TORC1), was observed to prevent TORC1 signaling during N deficiency, the activation of which contributes to the synthesis of proteins and, in turn, cell growth [20]. Among these activated or inactivated nutrient-signaling pathways during N deficiency, MAPK was the most well-known in higher plants; however, the number of mitogen-activated protein kinases in algae have never been reported. Based on the transcription results reported in our previous study [21], the aim of our study was to investigate the role of the MAPK signaling pathway in mediating 4 defenses against N deficiency in U. prolifera.

Plant materials and growth conditions
Algal materials of floating U. prolifera was collected from the coast of Qingdao (36°48'39.75''N; 121°38'10.88''E) on 10 th , May, 2018, which was approved by Qingdao Municipal Marine Development Bureau (Qingdao, China). Then the alga was pre-cultured at 20℃ in SPX-GB-250 intelligent illumination incubators (Botai, Shanghai, China) for 4 d in fresh distilled seawater supplemented with macro-elemental solution as previously reported [21], and the voucher specimens was maintained in fresh distilled seawater at 4℃. Ulva prolifera used for the further experiment was confirmed by both morphology and molecular analysis by Yi Yin according to the previous study [22,23]. The experiment was conducted according to the criterion of Convention on the Trade in Endangered Species of Wild Fauna and Flora.
After pre-cultural treatment, the U. prolifera was cultured in different medium created according to the study [24]. The composition for the pre-cultivation (N sufficient) or N-deficient medium consisted of the following: 450 mM NaCl 450 (pre-cultivation); 16.8 mM KNO 3 (pre-cultivation), 0 KNO 3 (N deficient); 0 KCl (pre-cultivation), 33.6 mM KCl (N deficient); 3.5 mM Na 2 SO 4 (pre-cultivation and N deficient); 100 mM 2-[4-(2-hydroxyethyl) piperazin-1-yl ethanesulfonic acid (HEPES); 5 mM with NaOH to 7.5. The high buffer capacity of the medium ensured that any change in pH value was never >0.6 units in the N-sufficient cultures, and that the pH did not change in N-deficient cultures.

Determination of growth rates and chlorophyll contents
Approximately 0.2 g U. prolifera was placed into 1-L glass flask in incubators and cultured in Nsufficient or N-deficient medium; each treatment was conducted in triplicate. At the indicated time point, the algal material was reweighed and the relative growth rates (RGR) were calculated as follows: RGR = (ln w2 − ln w1) / Δt, where w1 is the initial fresh weight and w2 the fresh weight after Δt days.
For chlorophyll detection, an approximately 0.1 g sample was ground in liquid nitrogen, weighed, and suspended in extraction buffer (80% v/v acetone). After incubation on ice for 15 min, the samples were centrifuged at 6000 rpm at 4℃ for 10 min. Chlorophyll concentrations (Chla and Chlb) were determined according to the methods described by Arnon [25]. The equations used were as follows: where V represents the volume of extraction buffer, and W represents the weight of the algal sample.

RNA extraction and quantitative reverse-transcription polymerase chain reaction analysis
Ulva prolifera was harvested at the indicated time point. After grinding in liquid nitrogen, RNA was extracted using TRIzol reagents (Thermo Fisher Scientific, Waltham, MA, USA). The RNA was purified using RNA resuspension buffer to remove any polysaccharides, as per the manufacture's protocol [26]. Two micrograms of RNA were reversed using the Hifair™ II 1st Strand cDNA Synthesis Kit (Yeasen, Shanghai, China). The program was set as 25℃ for 5 min, 42℃ for 60 min, and 85℃ for 5 min. Gene expressions were detected using the Hieff® qPCR SYBR Green Master Mix (Low Rox Plus) (Yeasen) and calculated as described by Liao et al. [27]. The specific primer pairs used for quantitative reverse-transcription polymerase chain reaction (qRT-PCR) are listed in Table 1; 18S rRNA was used as the internal controls. The 2 −ΔΔCT method [28] was used to analyze the expression levels of the indicated genes.

Full-length complementary DNA cloning and bioinformatics analysis
Because the sequence of UpNR from U. prolifera had been successfully cloned [29], we further designed primers for UpNiR and UpGOGAT based on the UniGene sequence from the transcriptome results of our previously study [21]; the primer pairs are listed in for 60 sec, and 72℃ for 3 min), followed by an extension reaction at 72℃ for 5 min. PCR products were gel-purified in 1% agarose gel, and the target fragment excised and cloned into a pESI-T vector (Yeasen). After transforming the fragments into competent Escherichia coli cells, positive recombinants were identified using PCR, and the clones were sequenced for verification (Invitrogen).
The UpNiR and UpGOGAT gene sequences were analyzed using the BLAST algorithm at the National To determine the viability of the cells, we used Evans Blue staining to estimate the percentage of viable protoplasts before or after exposure to N-deficient medium. For this, 1 μL 1% Evans blue was added to an aliquot of 25 μL protoplasts and incubated for 5 min at room temperature, after which the protoplasts were detected using a light microscope. The percentage of dead protoplasts that were stained with Evans Blue was calculated using the ratio of stained to total protoplasts.
To determine the percentage of protoplasts capable of regeneration, a known number of protoplasts were spread evenly over (or suspended in) a liquid medium in 24-well plates. Samples of regenerating protoplasts were centrifuged at 3,000 × g for 5 min and the resulting pellet suspended in a 0.1%

Statistical analyses
Results of all experiments are expressed as the means ± standard deviation (SD). P ≤ 0.05 was considered statistically significant. All statistical analyses were conducted using GraphPad Prism (https://www.graphpad.com/scientific-software/prism/).

N deficiency decreased the chlorophyll content and changes in wet weight
The growth ratio of U. prolifera in N-sufficient or N-deficient medium was observed for 6 d and the algae weights were recorded every 2 d. RGR of U. prolifera in N-deficient medium declined after the second day compared with that in the pre-cultivation medium (Figs. 1-A); however, N deficiency had no effect on the levels of Chla or Chlb, but the values of total chlorophyll decreased on the second day, which resulted from the decline trend of Chla and Chlb (Figs. 1-B, C, D).

UpNiR, and UpGOGAT sequence analyses
To verify the accuracy of sequences of UpNiR and UpGOGAT from the transcriptome results, we

UpNR, UpNiR, and UpGOGAT expression is induced by N deficiency
In higher plants and algae, NR synthesis is highly regulated, especially by sources of N, and our data demonstrated that the expression of UpNR, UpGOGAT, and UpNiR was stably maintained after exposure to N deficiency for 2 d; however, after 4 d., N deficiency induced the expression of UpNR and UpNiR, which suggested that both UpNR and UpNiR were the previous enzymes that responded to the N deficiency (Figs. 2-D, E). Hence, prolonged N deficiency led to significantly higher levels of UpGOGAT on day 6 (Figs. 2-F). All the data suggested that U. prolifera might resort to internal N sources, such as proteins and amino acids, to respond to abiotic stress; however, the mechanism by which this process is conducted in U. prolifera remains unclear.

N-deficiency induces protoplast apoptosis but does not reduce cell-wall regeneration
Most of the freshly isolated protoplasts were 7-15 and 30-40 μm in diameter and rich in pigment.
These protoplasts that were isolated using Fluorescent Brightener 28 staining showed no trace of blue fluorescence when examined under an ultraviolet florescence microscope, and dead protoplasts were stained dark blue with Evans Blue (Figs. 3).
To investigate the regeneration of protoplasts in different culture media, approximately 5 × 10 4 cells/well were plated onto 24-well plates and stained with Evans Blue and Fluorescent Brightener 28 for 1, 2, 3, or 4 d. The results showed that after incubating for 2 d, new cell walls began to synthesize and that the protoplasts cultured in N-deficient medium showed high mortality on day 4 followed by those exposed to N-sufficient medium (Figs. S1, Figs. 4-A). However, no significant changes in cellwall regeneration ratios were observed between N-sufficient and N-deficient medium, even in those protoplasts exposed to N-deficient stress for 4 d (Figs. S2, Figs. 4-B). Thus, we hypothesized that U.
prolifera might evolve some unique mechanisms by which to resist the stress caused by this nutrient deficiency.

Discussion
To our knowledge, N is the most important nutrient for plants and is an essential component of key macromolecules, including proteins and nucleic acids [31]. In addition, it is widely accepted that the N content in the Yellow Sea is essential for supporting the world's largest green tides [11,32]; therefore, it is critical that we identify the mechanism by which N triggers U. prolifera blooms. For prolifera has been minimal. In the present study, the full-length cDNAs of UpNiR and UpGOGAT were successfully cloned, and BLAST analysis confirmed that UpNiR and UpGOGAT were similar to the NiR from C. reinhardtii, and GOGAT from Auxenochlorella protothecoides, respectively. Hence, N deficiency induced the expression of UpNR and UpNiR in U. prolifera after exposure for 4 d and then reduced the expression to levels cultured in N-sufficient medium; however, the levels of UpGOGAT peaked at day 6. Based on these findings, we put forward the hypothesis that, in the initial 4 d, the increased UpNiR and UpNR might use proteins or amino acids as the N sources, then the metabolically produced ammonium is assimilated by UpGOGAT, which is why UpGOGAT peaked at day 6, after the occurrence of UpNR and UpNiR.
Hence, protoplasts were considered to generate when they could increase into a visible colony on an agar plate and then spread over the agar's surface. Successful regeneration required the use of a suitable inorganic salt, such as NaCl, MgSO 4 , and KCl, and N sources; therefore, the adaption of protoplasts in response to N deficiency could partly explain why U. prolifera maintained steady growth under fluctuating N sources. In this study, we successfully created protoplasts of different diameters, which began to form the cell wall after culturing for 2 d. Although N deficiency enhanced the mortality of the protoplasts compared with that in N-sufficient medium, but once the surviving protoplasts began to form a cell wall, the N nutrient had no effect on them. This might explain why N deficiency had no influence on cell-wall regeneration on the surface of protoplasts, even after 6 d.
It is widely accepted that N content in the Yellow Sea is essential to the support of the world's largest green tides [11,32]. Although the augments of UpNR, UpNiR, and UpGOGAT induced by N deficiency, as mentioned above, illustrated the potential possibility of the consumption of internal U. prolifera proteins as a defense against changing N contents within the environment, identifying the possible molecular mechanisms involved is also of great interest. It is well established that MAPK signaling mediates the process of cell growth and division in response to changes in environmental conditions, and exposure to the nutrient deficient environment leads to the activation of MAPK signaling in some plants [18]. Similarly, in our study, we found that N deficiency enhanced the levels of phosphorylated MAPK, and that this could be reversed by adding 10

Conclusions
This is the first report on a macroalgae that advances our understanding of the N-assimilation mechanism in Ulva and successfully cloned the N assimilation-associated enzymes. The activation of Declarations Ethics approval and consent to participate Not applicable.

Consent to publish
Not applicable.