2.1 Materials, handling and sampling
Lotus japonicus was planted in the artificial climate room of the Guizhou Prataculture Research Institute. The seeds were sown in 11*15 cm pots before planting, and the leaves were transplanted into bottles containing Hoagland nutrient solution with distilled water when 2–3 leaves had grown. The applied treatments were as follows: blank control (zl1) : 1/8-Hoagland nutrient solution, pH 8.1±0.5, with 10 mM NaHCO3 and 0.25 mM NH4H2PO4; low phosphorus (zl2): 1/8-Hoagland nutrient solution, pH 8.1±0.5, with 10 mM NaHCO3; and 20% drought stress (zl3): Hogland nutrient solution, pH 8.1±0.5, with 10 mM NaHCO3, 0.25 mM NH4H2PO4, and 200 g/L polyethyleneglycol (PEG-6000). Five seedlings were planted in each bottle, and the nutrient solution was changed every 3 days. The plants were harvested after one month of culture. Indicators were assessed, and samples were collected.
2.2 Transcriptome sequencing and quality control
Advanced molecular biology equipment was used to detect the purity, concentration and integrity of RNA samples to ensure the use of qualified samples for transcriptome sequencing. After the samples were qualified, a library was constructed: (1) eukaryotic mRNA was enriched with oligo (DT) magnetic beads; (2) mRNA was randomly interrupted by adding fragmentation buffer; (3) the first cDNA strand was synthesized with six-base random primers (random hexamers) using mRNA as a template, after which the second cDNA strand was synthesized by adding buffer, dNTPs, RNase H and DNA polymerase I, and the cDNA was purified with AMPure XP beads; (4) the purified double-stranded cDNA was subjected to terminal repair, a tail was added and sequenced, and fragment size selection was performed with AMPure XP beads; (5) finally, the cDNA library was enriched by PCR. After the construction of the library, the effective concentration of the library (library effective concentration > 2 nm) was accurately quantified by Q-PCR to ensure its quality. After passing the library inspection, different libraries were pooled according to the target amount of offline data and sequenced on the Illumina platform. The quality of the obtained data was strictly controlled, and the following filtering methods were applied: (1) removal of reads containing linkers; and (2) removal of low-quality reads (including reads with an N percentage greater than 10%, a quality value (Q) ≤ 10 or more than 50% alkali bases in the whole read). The high-quality clean data obtained after the above series of quality control steps were provided in FASTQ format.
2.3 Transcriptome data and bioinformatic analysis
The obtained clean data were compared with the reference genome of pulse roots by using HISAT2 [31]. The obtained mapped data were used for library quality evaluation, including insertion fragment length tests and randomness tests, and structure-level analyses such as alternative splicing analysis, new gene discovery and gene structure optimization were carried out [32]. Then, the StringTie maximum flow algorithm was to measure the levels of transcript or gene expression based on the index of fragments per kilobase of transcript per million fragments mapped (FPKM) values [33]. Differential expression analysis and the functional annotation and functional enrichment of differentially expressed genes were performed according to the expression levels of genes in different samples or different sample groups.
2.4 Metabolome sample processing and data analysis
To better understand the changes in different metabolites in the pulse roots under low-phosphorus stress and drought stress, three groups were selected for metabolite determination: the untreated control (zl1), low-phosphorus (zl2) and drought stress (zl3) groups; 6 samples were analyzed in each group, for a total of 18 samples. Metabolite extraction was performed according to the following step: weigh 50 mg of sample, add 1000 µL of an extract containing an internal standard (1000:2) (methanol;acetonitrile:water volumetric ratio = 2:2:1, internal standard concentration 2 mg/L), and perform vortex mixing for 30 s; add ceramic beads, grind at 45 Hz for 10 min, and perform ultrasonication for 10 min (ice water bath); leave standing at -20°C for one hour; centrifuge at 12,000 rpm at 4°C for 15 min; carefully remove 500 µL of the supernatant in an EP pipe; dry the extract in a vacuum concentrator; add 160 µL of extract (acetonitrile:water volumetric ratio = 1:1) to redissolve the dried metabolites; vortex for 30 s; perform ultrasonication in an ice water bath for 10 min; centrifuge the sample at 12,000 rpm at 4°C for 15 min; carefully remove 120 µL of the supernatant in a 2 ml injection bottle; and collect 10 µL of each sample mixed with the QC samples for analysis in a liquid chromatography-mass spectrometry (LC-MS) system. The LC-MS system used for metabolomic analysis was composed of a Waltz acquisition I-class plus ultrahigh-performance liquid chromatography (UHPLC) apparatus in series with a Waltz Xevo g2-xs QTOF high-resolution mass spectrometer. The chromatographic column was an acquisition UPLC HSS T3 chromatographic column (1.8 µm 2.1 * 100 mm) purchased from Waltz. The original data collected by Masslynx v4.2 were used for peak extraction, peak alignment and other data processing operations with Genesis Qi software. Identification was conducted based on the online METLIN database and the BmK self-built database of the Genesis Qi software. At the same time, the theoretical fragments were identified. The mass number deviation was within 100 ppm [34].
2.5 Proteome sample processing
To better understand the changes in pulse root proteins under low-phosphorus stress and drought stress, three groups were selected for metabolite determination: the untreated control (zl1), low-phosphorus (zl2) and drought stress (zl3) groups; three samples were selected for protein determination in each group, for a total of 9 samples. Quality control of protein extraction was performed according to the following steps: add 300 µL of 8 m urea (lysate: protease inhibitor 50:1); perform ultrasonication for 1 s followed by a 2 s hold for a total of 120 s; centrifuge at 4°C 14,000 g for 20 min; and perform protein quantification in the supernatant and SDS-PAGE quality control. Enzymatic desalting was conducted as follows: remove 100 µg of protein; add DTT at a final concentration of 10 mm; incubate at 37°C for 1 h; and restore to room temperature; add Iam at a final concentration of 40 mm; incubate away from light at room temperature for 45 min; dilute the sample with ammonium bicarbonate to pH = 8; add trypsin at a protein-to-trypsin ratio 50:1; incubate overnight at 37°C; the next day, add 50 µL 0.1% FA to terminate the reaction; desalt the sample with a C18 desalting column; activate the desalting column with 100% can; balance the column with 0.1% FA; load the sample onto the column; wash the column with 0.1% FA to remove impurities; finally, perform elution with 70% ACN, collect the flow through solution, and perform freeze-drying. Marking ot the samples was performed as follows: remove the TMT reagent; thaw it at room temperature; open the cover, and add 41 µL of acetonitrile; shake for 5 min and centrifuge the mixture; add 100 µG of TMT reagent to the enzyme-digested sample, and allow the reaction to occur at room temperature for 1 h; add ammonia to terminate the reaction; mix, vortex oscillate and centrifuge the labeled samples; dry the samples by centrifugal vacuum freezing; mix the labeled samples with 100 µL of mobile phase a to redissolve them; centrifuge at 14,000 g for 20 min; collect the supernatant; and analyze the samples by HPLC at a flow rate of 0.7 mL/min for classification.
2.6 Proteome LC-MS/MS
Proteome LC-MS/MS was performed as follows: prepare mobile phase liquid A (100% water; 0.1% formic acid) and liquid B (80% acetonitrile; 0.1% formic acid); dissolve the lyophilized powder with 10 µL of solution; centrifuge at 14,000 g at 4 °C for 20 min; collect 1 µ g of the supernatant for sample injection; and test the liquid quality. A Q Exactive HF-X mass spectrometer with a Nanospray Flex™ (NSI) ion source were used. The ion spray voltage was 2.4 kV; the ion transmission tube temperature was 275 degrees C; MS was conducted in data-dependent acquisition mode; the full scan range of MS was m/z 407–1500; the first-level mass spectrometer resolution was 60,000 (200 m/z); AGC was 3×106; and the maximum C-trap injection time was 20 ms. The parent ion with an ion strength in the top 40 in the full scan was selected and disintegrated by high-energy collision cracking (HCD) method for secondary MS detection. The resolution of secondary MS was set to 45,000 (200 m/z); AGC was 5×104; the maximum injection time was 86 ms; and the peptide fragmentation collision energy was set to 32% to generate the original MS data.