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 10th, 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 KNO3 (pre-cultivation), 0 KNO3 (N deficient); 0 KCl (pre-cultivation), 33.6 mM KCl (N deficient); 3.5 mM Na2SO4 (pre-cultivation and N deficient); 100 mM 2-[4- (2-hydroxyethyl) piperazin-1-yl ethanesulfonic acid (HEPES); 5 mM MgSO4·7H2O (pre-cultivation and N deficient); 2.5 mM CaCl2·2H2O (pre-cultivation and N deficient); 2.5 mM K2HPO4; 10 mM NaHCO3; 28 μM NaFe ethylenediaminetetraacetate (NaFeEDTA); 80 μM Na2EDTA·2H2O; 19 μM MnCl2·4H2O; 4 μM ZnSO4·7H2O; 1.2 μM CoCl2·6H2O; 1.3 μM CuSO4·5H2O; 0.1 μM Na2MoO4·2H2O; 0.1 μM Biotin, 3.7 μM vitamin B1; and 0.1 μM vitamin B12. The pH was adjusted 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 N-sufficient 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:
Chla = (12.71 × A663 - 2.59 × A645) × V / (W × 1000), and
Chlb = (22.88 × A645 – 4.67 × A663) × V / (W × 1000),
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.
Table 1 Primers for qRT-PCR and PCR analysis
Primers name
|
Sequences (5' to 3')
|
UpNR
|
Forward
|
CTGGATCGGTGGTCGTATGG
|
Reverse
|
AGCACCTTGTTGTCGTGGAA
|
UpNiR
|
Forward
|
CAACGTTCGCAACCTGACTG
|
Reverse
|
GCAGATGTTGATCTTGCGGG
|
UpGOGAT
|
Forward
|
TCTGCGACCTGCTATTGCTC
|
Reverse
|
AGCCGCTAGTCCACAAGAAC
|
18S rRNA
|
Forward
|
CGCAACTCCCGACTCACGAAGG
|
Reverse
|
ACCGGACCATGTGGCCCAGAAT
|
UpNiR-PCR
|
Forward
|
GGGGCTGATTGAAACGCAGTAG
|
Reverse
|
AACGGAGCCATCACATCACCC
|
UpGOGAT-PCR
|
Forward
|
CTAGGCGGACCTTTGCAGTGTTCTAT
|
Reverse
|
TCGAGCTTCTTCATCGGCGTGT
|
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 Table 1. The cDNA template was constructed as in previous studies, and partial sequences for these genes were obtained using 2 × Hieff Canace® PCR Master Mix (Yeasen). UpNiR and UpGOGAT PCR amplifications were conducted at 94℃ for 5 min, with 35 cycles (94℃ for 45 sec, 58℃ for 60 sec, and 72℃ for 90 sec). PCR amplifications for UpGOGAT were conducted at 94℃ for 5 min, with 35 cycles (94℃ for 45 sec, 58℃ 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 Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/blast). A phylogenetic tree was constructed using the neighbor-joining method with Mega 5.0 (http://www.megasoftware.net).
Protoplast isolation, Fluorescent Brightener 28 and Evans Blue staining
Approximately 1.0 g U. prolifera was cut and chopped into 0.5 - to 1.0-mm strips and immediately transferred into a deep petri dish containing WS1 buffer solution (0.5 M mannitol, 4 mM MES-KOH [pH 5.5], and 20 mM KCl). After shaking for 3 min, the liquid was removed, and 5 mL ES buffer (1.5% w/v cellulase R10, 0.4% w/v Macerozyme R10, 0.4 mM mannitol, 20 mM KCl, 20 mM 2-(N-morpholino) ethane sulfonic acid, pH 5.5 heated to 55℃ for 10 min and cooled to room temperature before adding 10 mM CaCl2 and 0.1% BSA). The algae were digested in ES buffer while shaking at 60-80 rpm for 5 h at 28℃. After enzymatic digestion, an equal volume of WS2 buffer (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, and 2 mM MES at pH 5.7) was added and the protoplast was filtered through 120-μm and 40-μm nylon mesh sieves. After configuring at 50 × g for 10 min, the protoplasts were washed twice with WS2 buffer, resuspended into the N-sufficient or N-deficient medium, and cultured separately in the dark for 24, 48, 72, and 96 h.
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% (w/v) solution of Fluorescent Brightener 28 (Sigma-Aldrich, St. Louis, MO, USA). After incubating for 5 min at room temperature, the samples were examined under oil using the Lecia fluorescence microscope according to the manufacture protocol [30].
Protein extraction and Western blot analysis
At the indicated time point, U. prolifera was harvested and ground in liquid nitrogen. The algal powder was put into a radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) with Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific) at 4℃ for 30 min. After centrifuging at 120,000 × g at 4℃ for 15 min, the suspension was collected and desaturated at 100℃ for 5 min.
Approximately 30 μg protein was separated with 12% sodium dodecyl sulfate–polyacrylamide electrophoresis gel and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocking in 5% nonfat milk diluted in TRIS-buffered saline and Tween 20 (TBST) for 1 h at room temperature, the membranes were incubated at 4℃ overnight with the following primary antibodies: phospho-p38 MAPK (Thr180/Tyr182) (3D7) rabbit mAb (dilution, 1/500; Cell Signaling Technology, Danvers, MA, USA), p38MAPK antibody (dilution, 1/500; Proteintech, Wuhan, China), β-actin antibody (dilution, 1/2500; PhytoAB, San Francisco, CA, USA). The membranes were then washed three times with TBST and the secondary antibodies (horseradish peroxidase conjugated goat anti-mouse IgG (H + L), or goat anti-mouse (H + L) purchased from Proteintech, Co., Ltd.) were laid separately on the membranes for 1 h at room temperature. Protein was detected using enhanced chemiluminescence reagents (Thermo Fisher Scientific), and analyzed using Image J (National Institute of Health, Bethesda, MD, USA); β-actin was used as the internal controls.
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/).