Plant materials and growth conditions
Experimental plots were conducted at the teaching and experimental farm of Yunnan Agricultural University in Kunming, Yunnan, China. P. notoginseng is a perennial herb; farmers have cultivated this medicinal crop for more than 400 years. One-year-old P. notoginseng seedlings were collected from the Wenshan Miao Xiang P. notoginseng Industrial Co., Ltd, China (Longitude 104°32 ', latitude 23°53 '). 1-year-old healthy rootstalks of P. notoginseng were selected in our experiments and transplanted to a white plastic pot (30 cm in diameter and 40 cm in depth) on January 2017, and 3 individuals per pot, 120 pots per treatment were arranged.
The soil had the following chemical characteristics: organic mater 0.573%, total N 0.201%, pH (H2O) 6.42, total phosphorus (P) 0.727 g/kg, ammonium N 39.93 mg/kg, available potassium (K) 0.019 mg/g, available P 4.88 mg/kg, soil water regime 12%. Pots were placed in environmentally controlled growth permeable black plastic net with growth irradiance of 10% full sunlight. Three N-fertilizer levels were applied in our experiments: (1) LN without N addition, (2) MN with 225 kg·ha-1 N addition in four applications, (3) HN with 450 kg·ha-1 N addition in four applications. N was supplied on April 22, June 22, July 22, August 22, 2017 respectively, along with 225 P2O5kg· ha-1 (superphosphate) and 450 kg·ha-1 K2O (potassium sulphate) in four applications.
P. notoginseng grown for eight months were used to determine plant mortality, leaf morphology and photosynthetic performance, and to collect leaves for comparative transcritome, chlorophyll and elemental N analyses. Five biological replicates were quickly frozen in liquid N and stored at -80 °C for RNA extraction.
Leaf anatomy and chloroplast ultrastructure
The juvenile leaves achieved for morphological and anatomical traits were used after 8 months of the N regimes. Leaf anatomical properties were performed in the method of paraffin section, and then the leaves were dehydrated in an alcohol series. Leaf tissues were embedded in paraffin (Thermo Scientific HistostarTM) and transversely sectioned at 10 mm thickness by means of microtome (Microm HM325, Walldorf, Germany). Finally, sections were stained with hematoxylin observed under a bright field Microscope (Zeiss Axio Cam HRC, Oberkochen, Germany).
A small piece of 1~2 mm2 was cut between the middle leaf vein and leaf edge and fixed with 2.5% glutaraldehyde and 1% osmic acid. According to the conventional series of ethanol dehydration, epoxy resin embedding, ultra-thin slicer sectioning, sectioning was stained with uranyl acetate and then stained with lead citrate, the chloroplast ultrastructure was observed under JEM100CX-II transmission electron microscope. For the estimation of Sc, 700 nm-thick sections were used by the method of Hanba et al [60].
Steady-state gas exchange rate
Steady-state gas exchange measurements were carried out using the photosynthesis system (Li-6400-40, Li-Cor, USA) with the 2 cm2 fluorescence leaf chamber. The leaf temperature and CO2 in the chamber were maintained at 25 °C and 400 mmol mol-1 during measurements, respectively. Subsequently, Photosynthetic light response curves and photosynthetic CO2 response curves were performed. Based on photosynthetic light response curves, full induction was complete, an automatic program of light response curves was run to measure the change in gas exchange rate with a set of PPFD. The level of PPFD was listed in the following order: 800, 500, 400, 300, 200, 100, 80, 60, 40, 20 and 0 mmol m-2s-1, each light intensity stabilized for 5 min. The relationship between Anet and PPFD was fitted, Anet= Amax - AmaxC0e-αPPFD/ Amax, where Amax is the maximum net photosynthetic assimilation under saturating light, α is the apparent quantum efficiency (AQY), where AQY was estimated by the slope of the linear region of the light response curve. C0 is the index to measure the net photosynthetic rate approaching 0 in low light. According to the parameters in the formula, dark respiration rate (Rd) = Amax- AmaxC0.
Anet and Ci were evaluated at a range of reference CO2 concentrations (400, 300, 200, 150, 100, 50, 400, 600, 800, 1000 and 1200 1500 mmol mol-1). CO2 response curves and CE were achieved by fitting the data to a nonrectangular hyperbola and the slope the linear region of the CO2 response curve, respectively. Vcmax and Jmax was gained according to the idea offered by Buckley and Diazespejo [108], this calibration requires measurements under low O2.
Chlorophyll fluorescence of PSII
At predawn, minimum and maximum Chl fluorescence yield (FO and Fm) was measured in the fully dark-adapted leaves. Minimum, maximum and steady-state fluorescence intensity (FO`, FV`, Fm` and Fs) were made in the process of light response curves. Fv`/Fm` was estimated as (Fm` – FO`)/ Fm`; FPSII as (Fm`– Fs)/Fm`; JT= PPFD×FPSII×aleaf×b, commercial fluorometers usually provide an estimate of PSII total electron transport rate (JT) by assuming that 400-700 nm (PAR) leaf absorptance (aleaf) equals 0.84 [109] and that absorbed photons (b) are equally distributed between the two photosystems (b=0.5)[110]. This approximation is reasonable for comparison of JT between optically similar samples such as leaves of cultivars of a single plant species[111]. Moreover, there was a curvilinear relationship between aleaf and chlorophyll content, whereas the curvature was extremely low when the chlorophyll content was > 0.4 mmol m-2 [30, 112]. According to Evans and Poorter [112], the calculation of aleaf demonstrated that aleaf (0.84, 0.85, and 0.85, in leaves with low, moderate, and high N content, respectively) was similar to the value of 0.84 [113-115]. Therefore, in this study, aleaf also assumed to be 0.84, and b was assumed to be 0.5 [110, 116]. NPQ as (Fm – Fm`)/Fm`, and qP as (Fm` – Fs)/(Fm` – F0`). Jc and Jo was calculated according to the method of Valentini et al [117], JO = 2/3×(JT − 4×(Anet + Rd)) , JC = 1/3×(JT + 8×(Anet + Rd)). According to the methods of Manter and Kerrigan [118], gm and Clip were calculated as gm=Anet/{Ci-Γ*×[JT+8(Anet+Rd)]/[ JT-4(Anet+ Rd)]}, Clip=Γ*×[JT+8(Anet+Rd)]/[ JT-4(Anet+ Rd)], where Γ* is the CO2 compensation point. Cc was calculated as Cc= Ci × S*/S. The initial slope of the regression of Jc/Jo to Ci/O was used to S*(Additional file 12: Figure S10), O2 concentration (210 mmol CO2 mol-1). S was calculated as follow: S=O/2Γ*. glip can be showed that glip= Clip ×Sc. gi was calculated by gi= Amax/(Ci-Cc) .
Calculation of N allocation in photosynthetic components
Leaf N was determined with Kjeldahl. SLN was calculated. Photosynthetic-related pigments were determined by the method of Xu et al. [92] and Thayer & Björkman [119]. NC, NB and NL were determined according to the method of Niinemets et al [120]. Nphoto is the sum of NC, NB and NL. PNUE is the ratio of leaf N used for C fixation per unit leaf area. The formula is as follows:
NC=[Vcmax/(6.25 ×Vcr ×SLN)]×SLN (1)
NB=[ Jmax/(8.06 ×Jmc×SLN)] ×SLN (2)
NL=[Cc/(CB×SLN)] ×SLN (3)
Nphoto=NC+NB+NL (4)
PNUE=Amax/SLN (5)
Vcr is the Rubisco specific activity with a value of 20.8 mmol CO2·g-1 Rubisco·s-1. Jmc is the maximum electron transfer rate per unit cytochrome f (Cyt f) with a value of 155.6 mmol electrons·mmol-1 Cyt f·s-1. Cc is the leaf chlorophyll content (mmol·m-2), CB is the combined light system I (PSI), photosystem II (the chlorophyll in PSII) and PSII light-harvesting pigment complex (LHCII) with a value of 2.15 mmol·g-1 N.
Leaf Rubisco content and activity
The Rubisco content was determined according to Makino et al [121]. Briefly, newly expanded leaves were stored at -80℃. 0.5 g frozen leaves were ground in a solution containing 50 mM Tris-HCl (pH =8.0), 5 mM b-mercaptoethanol, and 12.5% glycerol (v/v), and then centrifuged at 1500 g for 15 min at 4℃. The supernatants were mixed with a solution containing 2% (w/v) SDS, 4% (v/v) b-mercaptoethanol and 10% (v/v) glycerol, boiled in a water bath for 5 min before SDS-PAGE using a 4% (w/v) stacking gel, and a 12.5% (w/v) separating gel. After electrophoresis, the gels were stained with 0.25% Commassie Blue for 12 h, and destained. Gel slices containing the large subunits and small subunits of Rubisco were transferred to a 10 mL cuvette containing 2 ml of formamide and incubated at 50℃ in a water bath for 6h. The absorbance of the wash solution was measured at 595 nm. Protein concentrations were determined using bovine serum albumin as a standard. Bovine serum albumin (BSA) was measured at 595 nm as standard protein.
Rubisco activity was measured according to Parry et al [122] with minor modifications. The extraction solution contained: 50 mM Tris-HCl (pH=7.5), 10 mM b-mercaptoethanol, 12.5% (v/v) glycerol, 1 mM EDTA-Na2, 10 mM MgCl2 and 1% (m/v) PVP-40. Extracts were clarified by centrifugation (8000 g at 4 ℃ for 10 min) and the supernatant was immediately assayed for Rubisco activity.
RNA extraction and Library construction, sequencing
RNA samples were extracted using RNA pre-pure Plant Kit (Tiangen, Beijing, China). After total RNA was extracted, mRNA was enriched by Oligo (dT) bads, and then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. The cDNA fragments were purified with QiaQuick PCR extraction kit, end repaired, poly(A) added, and ligated to Illumina sequencing adapters.The ligation products were selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina HiSeqTM 4000 by Gene Denovo Biotechnology Co. (Guangzhou, China).
Raw reads filtering and de novo assembly
Low quality reads containing adapters, more than 10% of unknown nucleotides (N), were eliminated. Transcriptome de novo assembly was carried out with short reads assembling program-Trinity. The redundancy was eliminated by the TGICL software and further assembled into a set of non-redundant unigenes.
105G sequencing data were obtained and de novo assembled into 93162 unigenes (Additional file13: Table S3) with an average length of 790 bp (Additional file 14: Table S4). Collectively, 41569 (44.62%) unigenes were functionally annotated in accordance with their parallels with known genes/proteins in the databases. The particular statistics of the functional annotation are emerged as in Additional file 15: Figure S11. After eliminating adaptors, unknown nucleotides and low quality reads, the data generated 43588606, 46978940, 43177242 paired-end 125-bP reads in the LN, MN and HN treatments, respectively, coinciding with approximately 6.48 Gb data (Additional file16: Table S5) .Q20 percentages exceeded 98%, uncalled base (“N”) percentage was equal to 0% per sample (Additional file 16: Table S5). The GC contents were almost identical for all 15 leaves tissues, ranging from 43.08 to 44.20% (Additional file 16: Table S5). In general, between 83.23% and 84.79% of clean reads could be mapped on full gene set (Additional file 17: Table S6). A Pearson’s correlation analysis revealed high correlations between biological replicates (R2 =0.8671 to 0.9769, Additional file18: Figure. S12).
Basic annotation of unigenes
To annotate the unigenes, we used BLASTx program (http://www.ncbi.nlm.nih.gov/BLAST/) with an E-value threshold of 1e-5 to NCBI non-redundant protein (Nr) database (http://www.ncbi.nlm.nih.gov), the Swiss-Prot protein database (http://www.expasy.ch/sprot), the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg), and the COG/KOG database (http://www.ncbi.nlm.nih.gov/COG). Protein functional annotations are obtained according to the best alignment results.
Analysis of DEGs
To identify DEGs within N regimes, the normalized read counts from five replicates of each sample were analyzed and the edge R package (http://www.r-project.org) was used. We identified genes with a fold change ≥2 and a false discovery rate (FDR) <0.05 in a comparison as significant DEGs. DEGs were then subjected to enrichment analysis of GO functions and KEGG pathways.
GO enrichment analysis and pathway enrichment analysis
All DEGs were mapped to GO terms in the Gene Ontology database (http://www.geneontology.org), gene numbers were calculated for each term, significantly enriched GO terms in DEGs comparing to genome background were defined by hyper geometric test. KEGG enrichment analysis was carried out through Genomes database (g" http://www.genome.jp/kegg). P-value of GO terms and KEGG pathway was gone through FDR Correction, taking FDR ≤ 0.05 as a threshold.
RT-qPCR assay
To validate the expression of 19 significant DEGs observed in RNA-Seq data, reaction was carried out using EvaGreen 2X qPCR MasterMix Kit (abm, Vancouver, Canada) in a QuanstudioTM 5 Real-Time PCR Intruments (Thermo Fisher Scientific, Inc.). First-strand cDNA was synthesized using the RevertAidTMFirst strand cDNA Synthesis Kit (TransGen Biotech, Beijing, China). DEGs primers were designed using the Primer-Blast (/" https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and synthesized commercially (Shuoqing, Kunming, China). Actin were selected as reference genes [123].The primers used in qRT-PCR analyses are listed in Table S1. Amplification reaction mixtures were made of 10 µL of Eva Green 2X qPCR Master Mix, 0.5 µL of each forward and reverse primer (10 mM), and 1 µL of cDNA template, and ddH2O was added to a final volume of 20 µL. The amplification cycling program was as follows: enzyme activation was operated at 95 ℃ for 10 mins, moreover, 40 cycles of 95 °C for 15 s, 58 °C for 30 s and 72 °C for 30 s. The results were analyzed using the software accompanying the QuanstudioTM 5 Real-Time PCR instruments. The relative expression values were obtained by using the 2-ΔΔCt method [124].
Statistical analyses
Statistical analyses were performed with SPSS software package (Chicago, IL, USA) and SigmaPlot 10.0, where the data were tested to confirm their normality and the variables were present as the mean ± SD (n=5-7). We obtained 7 repetiotions that studied physiological parameter for N- cultivated plants, and we generally obtained 5 repetions for bioinformatic analyse. Differences were considered significant when P < 0.05 according to the ANOVA F-test. The Ct values derived from qPCR were normalized and the relative fold changes in transcripts were calculated using the relative expression software tool, REST.