Plant growth
The lotus seed species, Taikong36, was selected for all experiments in this study. The seeds used in this study were derived from the open field of Yangzhou University, and necessary permission was obtained to collect these samples. In spring (usually in April), the lotus was planted in the field for germination with moist soil. After the petiole broke through the soil surface, the water depth was maintained at 5-10 cm; a deeper water (20-40 cm) was required for the development of the plant and temperature was maintained between 20 and 30 °C during the entire growth season. The application of fertilizer and pest control was carried out in the same manner employed in the conventional management of the field. The seeds were harvested in November and placed in a container at normal temperature.
The role of sucrose in AR formation
The seed coat of lotus was punched to enable the uptake of available water (for approximately three days) and placed at 26 °C for approximately three days under dark conditions. Fifty germinated seedlings from each treatment were selected and placed into 0 g/L, 20 g/L, and 50 g/L sucrose solutions for two days. The germination rates and numbers were counted on days 0, 2, 4, and 6 after transfer into water for 2 d. All statistical data were expressed as the mean ± SE of three repetitions of experiments. The SPSS software ver. 14.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analyses.
Observation of the paraffin sections
The lotus seed coat was broken and immersed in water for approximately three days for germination at 26 °C in the dark. The germinated seeds were selected for treatment with various concentrations of sucrose (0, 20, and 50 mg/L) for two days. The seedling hypocotyls of plants subjected to the above treatments were selected from four time points (0, 2, 4, and 6 d) after treatment. The selected samples were cut into small pieces of 2.5 mm x 2.5 mm x 2 mm (length, width and height), and then placed in a container filled with FAA fixing fluid (solution amount was 20-fold that of samples).
The container with fixed samples was produced in a vacuum state using a syringe for 5 s. The lid of the container was opened for gas exchange after five min. This process was repeated in triplicate. The container was transferred to a clean bench at normal temperature for 24 h. Thereafter, 50%, 70%, 85%, 95%, and 100% ethanol were applied to dehydrate the samples for approximately 25-30 min. After dehydration with ethanol, the samples were treated with a mixed solution (one-half volume of ethanol and one-half volume of xylene and pure xylene) for approximately 25-30 min. Paraffin debris was prepared and thawed on an electric stove and then poured into the container with the samples for 24 h. The tissues embedded in paraffin were cut into small blocks, and 10 µm of wax tape was prepared using a slicer. The wax tapes were placed on a glass slide, and then respectively transferred into pure xylene, mixed solution (one-half volume of pure xylene and one-half volume of absolute ethanol), and absolute ethanol for 10 min. The slide was dried at room temperature, and 2-3 drops of 0.75% crystal violet solution were placed on the samples for 5 min. The samples were washed with distilled water and then observed using an optical microscope.
Determination of monomer lignin and polymer lignin contents
The seed coat was broken for water absorption. The broken seeds were immersed in water (26 °C in the dark) for three days. After germination, two concentrations (20 mg/L and 50 mg/L) of sucrose were applied as treatments to the plants for two days, with water as a control. The hypocotyls of seedlings were used as materials to determine the monomer lignin and polymer lignin contents. Ten milligrams of dried sample was added to a mixture (2.5% boron trifluoride, 10% ethanethiol, 87.5% dioxane) for 4 h in a metal bath followed by 300 µL of 0.4 m sodium bicarbonate. The mixture was vibrated, and 2 ml water and 0.3 ml ethyl acetate were added. The mixture was centrifuged at 14000 rpm for 10 min at normal temperature, and the supernatant was retrieved. The supernatant was dried with nitrogen, and 150 µL pyridine (including an internal standard) and 50 µL trimethylsilane were allowed to react at 60 °C for 1 h. The mixture was centrifuged at 10000 rpm for 10 min at normal temperature, and the supernatant was retrieved for lignin identification. The lignin content was determined using a GC-MS instrument (7820A-5977B) from Agilent Technologies Inc. (CA, USA). The conditions for chromatography (mass spectrometry) and quantitative analysis lignin were based on a previous protocol [31]. Three biological replicates was carried out in this experiment. In addition, the roots of five- to six-leaf stage seedlings of transgenic Arabidopsis plants and none of the transgenic plants were collected for polymer lignin identification. The material treatment and identification method was the same as that mentioned above.
Metabolite analysis of lignin during AR formation
Lotus seedlings were treated with sucrose (20 g/L and 50 g/L) for two days, the detail progress were the same as above mentioned. The hypocotyls of control plants and plants treated with 20 g/L and 50 g/L were selected for metabolites analysis. All freeze-dried hypocotyls were crushed using a mixer mill (MM 400, Retsch) with a zirconia bead for 1.5 min at 30 Hz. Powder powder (100 mg) was weighed and extracted overnight at 4 °C with 1.2 ml 70% aqueous methanol. Following centrifugation at 10,000 g for 10 min at normal temperature, the extracts were absorbed (CNWBOND Carbon-GCB SPE Cartridge, 250 mg, 3 ml; ANPEL, Shanghai, China, www.anpel.com.cn/cnw) and filtered (SCAA-104, 0.22μm pore size; ANPEL, Shanghai, China, http://www.anpel.com.cn/) before UPLC-MS/MS analysis.
UPLC conditions
The sample extracts were analyzed using a UPLC-ESI-MS/MS system (UPLC, Shim-pack UFLC SHIMADZUCBM 30A system, www. shimadzu. com.cn/; MS, Applied Biosystems 6500QTRAP, www.appliedbiosystems.com.cn). The analytical conditions were as follows: column, Waters ACQUITYUPLCHSST3C18 (1.8 µm, 2.1 mm*100 mm); and mobile phase, solvent A- pure water with 0.04% acetic acid and solvent B- acetonitrile with 0.04% acetic acid. Sample measurements were performed with an Agilent program that employed the starting conditions of 95% A and 5% B. Within 10 min, a linear gradient to 5% A and 95% B was employed, and a composition of 5% A and 95% B was maintained for 1 min. Subsequently, a composition of 95% A and 5.0 % B was employed for 0.10 min and retained for 2.9 min. The column oven temperature was set to 40 °C, and the injection volume was 2 μL. The effluent was alternatively connected to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS.
ESI-QTRAP-MS/MS
LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (Q TRAP), API 6500 Q TRAP UPLC/MS/MS System, equipped with an ESI Turbo Ion Spray interface, operating in positive and negative ion mode and controlled by Analyst 1.6.3 software (AB Sciex). The ESI source parameters were as follows: ion source, turbo spray; source temperature, 550 °C; ion spray voltage (IS), 5500 V (positive ion mode)/-4500 V (negative ion mode); ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) 50, 60, and 30.0 psi, respectively; and collision gas (CAD), high. Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in the QQQ and LIT modes, respectively. QQQ scans were acquired as MRM experiments with a collision gas (nitrogen) set at 5 psi. DP and CE for individual MRM transitions were performed with further optimization of DP and CE. A specific set of MRM transitions was monitored for each period, according to the metabolites eluted within this period.
RNA-seq analysis of the differentially expressed genes
For the analysis of differentially expressed genes, all genes related to monomer and polymer lignin synthesis were selected and their expression profiles were monitored after treatment with sucrose during ARs formation. The lotus seed coat was broken before germination and placed in 5 cm water at 28-30 °C for germination. After germination, the seedlings were treated with sucrose (20 mg/L and 50 mg/L) and IAA (10 μM and 50 μM) for two days, and then placed in water for continued growth. RNAs were then extracted, and eight libraries were constructed for gene expression analysis (low concentrations of sucrose and IAA were demonstrated to promote AR formation while high concentration inhibited AR development in lotus seedlings). In the previous study, four libraries treated with different concentration of IAA (CK library: germinating seeds without treatment; A library: initial AR stages: 2 days of seedlings with 10 μM IAA treatment; B library: 2 days of seedlings without IAA treatment; C library: 2 days of seedlings with 50 μM IAA treatment) and sucrose (C0 library: the samples were collected at day 0; CK1, GL20 and GL50 libraries: the samples were collected at day 1) were constructed, and the data has been published by Cheng et al. [32] and Cheng et al. [20] respectively. Gene expression related to the synthesis of lignin including monomer and polymer lignin was selected from above libraries for further analysis.
Cloning and sequence analysis of NnLAC17
The sequence of NnLAC17 was derived from the NCBI database according to the gene expression profile under sucrose treatment. Lotus RNA was extracted from the hypocotyls of three old seedlings using a plant RNA extract mix (Tiangen, Beijing, China). DNase was applied to eliminate residual DNA before cDNA synthesis, which was performed according to the protocol of the mix kit (Promega, USA). A total of 20 μL of PCR reaction mixture, which included 2.5 μL dNTP, 2 μL forward and reverse primers, 2.5 μL MgCl2, 0.5 μL Taq polymerase, 2 μL cDNA fragments, and 9.5 μL dH2O, was used. The PCR program included 35 cycles: 94 °C for 1 min, 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min, and 72 °C for 10 min. Forward primer: 5-ATGGGTTCCTTTGTTCTTCC-3; Reverse primer: 5-AGGCGGTAGTTTCTGATTTG-3. The plasma of DNA plasid was sent to Sangon Biotechnology Co., Ltd (Shanghai, China) for sequencing. For sequence analysis, five LACs including lotus, vitis riparia, vitis vinifera, spatholobus suberectus, and trema orientale were used for alignment anaysis by DNAman software. In addition, thirty-four LACs from different plants were selected, and MEGA-X software was used for phyogentic tree construction.
Expression analysis of NnLAC17 in lotus
The seed of the lotus was placed in a glass container with a water depth of 5 cm to allow germination at 28-30 °C. The germinated seeds were treated with sucrose (20 mg/L and 50 mg/L) and IAA (10 μM and 150 μM) for 2 d and transferred into water to allow growth. The expression profile of NnLAC17 was determined using qRT-PCR. The treatment of lotus seeds and the germination conditions were the same as those mentioned above. A total of 150 two-leaf seedlings which were divided into three groups, were treated with various concentrations of sucrose (20 g/L and 50 g/L) and IAA (10 μM and 150 μM) for 3 days respectively, and then transferred to water for continued growth. The hypocotyls of seedlings at 4 d after treatment were selected for transcription-level analysis. For the expression of NnLAC17 in different organs, the leaf, stem, and hypocotyls of seedlings at 6 d (cultivation in water) were selected to monitor expression changes. Total plant RNA was derived from these samples, and DNase was used to eliminate residual DNA. The primer was designed based on the NnLAC17 sequence using the primer 5.0 software. Forward primer was 5-GGGTTCCTTTGTTCTTCCA-3, and reverse primer was: 5-GCCAGTGCAAGGTGATATT-3. NnActin was used as the internal standard. The NnActin forward primer was 5'-ACGCGTATGAAGTCAGTTGT-3' and reverse primer, 5'-TTTATGGGGATCAGCTGGT-3'. A 25 μL reaction mixture was prepared, which contained 12.5 μL SYBR Premix Ex Taq II (Tli RNaseH Plus) (2×), 1 μL of each of the forward and reverse primers, 2 μL cDNA, and 8.5 μL dH2O. The PCR program consisted of 30 s at 94 °C, followed by 40 cycles of 95 °C for 5 s and 60 s at 60 °C. Three biological replicates was carried out in this experiment. For data analysis, the 2 -△△Ct method was used to identify NnLAC17 expression. The △Ct value was obtained according to the Ct (target) and Ct (actin) values in treated plants (△Ct (target)) and control (△Ct (normal), and the △△Ct value was calculated according to the data of △Ct (target) and △Ct (normal). Based on the △△Ct value, the 2-△△Ct value was determined.
Vector construction
NnLAC17 was cloned and inserted into a clone vector (pGEM-T). pGEM-T was transformed into Escherichia coli to expand reproduction. After identification, positive clones were cultured in LB culture medium. The plasmid was extracted from bacterial fluid and digested with BamHI and KpnI enzymes. Thereafter, the digestion product (sequence of NnLAC17) was inserted into the plant transformation vector with a CaMV 35S promoter. The pSN1301:: NnLAC17 plasmid was inserted into the Agrobacterium tumefaciens strain, GV3101. The floral dip method [33] was employed to transform NnLAC17 into the wild-type Arabidopsis plant. The transformed Arabidopsis was cultured in the greenhouse, and seeds were harvested after approximately three months of growth. Sterilized seeds of the T0 generation were tiled on MS medium containing 20 μg·g−1 hygromycin B to select ‘positive’ plants. The temperature of the chamber was 22 °C with 12 h light and 12/dark light cycle. In addition, RT-PCR method was applied for further identification, and the primers was: Forward primer was 5-GGGTTCCTTTGTTCTTCCA-3, and reverse primer was: 5-GCCAGTGCAAGGTGATATT-3. Reaction mixture and program process were the same as gene expression. Base on the information of data, more than five lines of positive plants were selected for further study.
Identification of NnLAC17 function in Arabidopsis plants
The seeds of transgene (T2 generation) and wild-type plants were cultured in pots, and six-leaf-old transgenic seedlings and wild-type plants were selected for functional analysis. The development of all emerged roots including root length and number were determined. For root number analysis, all the root including ARs, primary root and lateral root were together counted (the hypocotyl of Arabidopsis was too short, so it was difficult to clearly distinguish ARs and primary root. Therefore, all the roots were identified together). For statistical analysis, all data are presented as the mean ± SE of triplicate independent treatments with approximately 10 seedlings per experiment, and about thirty seedlings was used for three biological replicates. The SPSS software ver. 14.0 (SPSS Inc., Chicago, IL, USA) was for data analysis.