Plant materials
The seeds of the recalcitrant Malaysian rice cultivar MR 219 used in this research were obtained from Malaysian Agricultural Research and Development Institute (MARDI), Seberang Prai, Penang.
Lignosulfonates preparation
Analytical grade NaLS (471038; Sigma-Aldrich, USA) and CaLS (471054; Sigma-Aldrich, USA) were prepared in a stock solutions of 50 mg/mL. All the stock solutions were filtered sterilized using 0.22 μm cellulose acetate membrane before being kept at 4 ºC.
Seeds sterilization and LS treatment
Surface sterilization were performed on mature seeds according to previously described protocol37 with slight modifications. Firstly, the seeds were de-husked and sterilized with 70% (v/v) ethanol for 1 min, followed by 50% (v/v) Clorox for 30 min. The seeds were rinsed with sterile distilled water before being dried on filter paper. The sterilized seeds were then transferred into our previously established callus induction medium containing Gamborg’s B5 basal medium38 supplemented with 10 g/L maltose, 0.1 g/L L-glutamine, 0.1 g/L L-asparagine, 0.1 g/L L-arginine, 10 mg/L NAA and 1 mg/L 2,4-D, pH 5.811. The seeds were then incubated under a photoperiod of 16 h light and 8 h darkness at 25 ± 2 ºC. After one week, approximately 1 cm of the shoot apices were removed from the seed and cultured in shoot growth medium containing Murashige and Skoog medium (MS)39 with 30 g/L sucrose, 3 mg/L kinetin and 0.5 mg/L NAA supplemented with CaLS or NaLS at different concentrations (100, 200, 300, and 400 mg/L). The shoot apices were incubated under a photoperiod of 16 h light and 8 h darkness at 25 ± 2 ºC for three weeks.
Protein extraction and protein digestion
In proteomic analysis, plant samples were ground into fine powder using liquid nitrogen. The samples were then mixed with 500 μL protein extraction buffer containing 50 mM of ammonium bicarbonate and 10 mM phenylmethylsulfonyl fluoride (PMSF). The mixture was then vortexed, sonicated and centrifuged according to Yang et al.40 and solubilized protein was collected. Desalting was carried out on total soluble protein obtained using acetone precipitation method41. Subsequently, the proteins content in the plant sample was determined at 595 nm through Bradford assay42. The proteins sample (100 μg) was digested with Trypsin Gold (Promega, USA) at a ratio of 1:200 parts of protein, according to previous study40.
Peptide separation and protein identification
Nano liquid chromatography tandem-mass spectrometry (nanoLC-MS/MS; Thermo Scientific, USA) analysis was performed according to Yang et al.40. Briefly, aliquot of 2 μL digested sample was injected into EASY-Spray Column Acclaim PepMapTM C18 100 (A0, 2µm particle size, 50µm id x 15cm) at 35 ˚C. The sample elution process was performed similarly as described by Yang et al.40. The eluents from the LC were directly introduced into a mass spectrometer (Orbitrap Fusion – Thermo Fisher Scientific, US). The instrument was operated in the data dependent acquisition. Full scan spectra were collected (OTMS1) using parameters defined by previous study40. Only precursors with an assigned monoisotopic m/z and a charge state up to 4 were further analyzed for MS2. All precursors were filtered using a 20 sec dynamic exclusion window and intensity threshold of 5,000. The MS2 spectra were analyses (ITMS2) following parameters reported by Yang et al.40. Precursors were fragmented by CID and HCD at normalized collision energy of 30% and 28%, respectively.
Subsequently, raw data was analyzed using Thermo ScientificTM Proteome DiscovererTM Software 2.1 and SEQUEST HT was used as the database searching algorithm. The intensities of each MS ion were measured according to the accurate mass and time tag strategy43. Identification of the proteins was performed based on the searched against the UniprotKB database restricted to O. sativa (2020_01: 48,904 sequences) with a 1% strict FDR and 5% relax FDR criteria using Percolator®. Search parameters were set up according to previous study40.
Protein quantification and data analysis
The experiment was performed in triplicates with three biological replicates for MSO and NaLS-treated samples. The protein files (txt.format) obtained from Proteome DiscovererTM were uploaded to Perseus for comparative proteome analysis between MSO and NaLS-treated samples. Data processing such as PCA and Pearson correlation was performed in Perseus according to Ramdas et al.43. Significant differences of protein abundance were determined based on the Student’s t test (p < 0.05). The p-values were also adjusted for multiple-testing using the permutation-based false discovery rate, with a randomization number of 250. Proteins were considered to be significantly differentially expressed between treatment groups with adjusted p-values of <0.05.
Total photosynthetic pigments content
One hundred milligram of sample was ground into fine powder with the presence of liquid nitrogen. The ground powder was mixed with 2 mL of 80% (v/v) of acetone for 1 min in darkness. The homogenate was then centrifuged at 400 x g for 5 mins. The absorbance of the samples was then recorded at 470, 647 and 663 nm. The concentration of photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll and carotenoids) were calculated according to calculations described in Lichtenthaler et al.44 and expressed in mg/g fresh weight (FW).
Rubisco activity
Rubisco activity was measured spectrophotometrically according to Usuda45 with slight modifications. In brief, 1 g of leaf sample was homogenized to fine powder with presence of liquid nitrogen. Then, the powder was mixed with ice-cold extraction buffer containing 0.25 M Tris-HCl (pH 7.8), 0.05 M MgCl2, 0.0025 M EDTA and 37.5 mg of DTT. Centrifugation of 10,000 x g for 10 minutes at 4°C was performed and the supernatant was collected as crude enzyme. Approximately 40 µL of crude enzyme was mixed with 960 µL reaction buffer containing 100 mM Tris-HCl (pH8), 40 mM NaHCO3, 10 mM MgCl2, 0.2 mM NADH, 4 mM ATP, 0.2 mM EDTA, 5 mM DTT, 1 U of glyceraldehyde-3-phosphodehydrogenase, 1 U of 3-phosphoglycerate kinase, and 0.2 mM ribulose 1,5-bisphosphate (RuBP). The absorbance of enzyme activities was recorded at 340 nm and expressed in µmol CO2 mg−1 protein.
Total soluble sugar content
Total soluble sugar content was estimated according to Dubois et al.46 with slight modifications. In brief, 0.1 g of sample was ground into powder in liquid nitrogen and extracted twice in 2 mL of 90% (v/v) ethanol at 60°C for one hour. After each extraction, samples were centrifuged at 400 x g for 5 mins. One mL of the supernatant was mixed with 1 mL of 5% (v/v) phenol together with 5 mL of concentrated sulphuric acid. The mixture was allowed to cool before the readings was taken at 495nm spectrophotometrically. The soluble sugar content was determined using glucose as a standard and expressed in mg/g FW.
Total protein content
Two hundred fifty milligram of plant sample were ground into powder with liquid nitrogen. Ice cold extraction buffer containing 1.8 mL of 50 mM ammonium bicarbonate (ABC) and 0.2 mL of 100 mM PMSF was added to the powdered sample. The homogenate was centrifuged at 10,000 x g for 30 mins and supernatant was collected as crude enzyme. All steps in enzyme extraction was performed at 4°C. Bradford assay was performed to determine the protein concentration at 595 nm42. Total protein content in the sample was then determined by using bovine serum albumin as standard.
Peroxidase activity
The crude enzyme obtained during protein extraction was used in determination of peroxidase activity. One hundred microliters of crude enzyme were added with 950 µL of distilled water, 750 µL of 100 mM potassium phosphate buffer (pH 6.8), 600 µL of 100 mM pyrogallol and 600 µL of 100 mM H2O2. Peroxidase activity was measured at 420 nm between the second and fifth minute after addition of crude enzyme into the substrate. The peroxidase activity was expressed as micromoles of purpurogallin formed per minute per milligram of proteins47
Malondialdehyde (MDA) content
MDA content was measured according to Luo et al.48 with slight modifications. Approximate 0.2 g of frozen powdered samples were dissolved in 10 mL of 10% (w/v) trichloroacetic acid (TCA). The homogenate was then centrifuged at 12,000 x g for 10 mins. Subsequently, 2 mL of supernatant was mixed with 2 mL of 10% (w/v) TCA containing 0.6% (w/v) of thiobarbituric acid (TBA) and incubated at 100°C for 20 mins. Then, the homogenate was allowed to cool on ice followed by centrifugation at 12,000 x g for 10 mins. The supernatant was then measured at 532, 600 and 450 nm. The MDA content was calculated using the following formula; MDA content (uM) = 6.45 (OD532−OD600) − 0.56 (OD450).
Phenylalanine ammonia-lyases (PAL) activity
PAL activity was measured according to Schmidt et al.49 with slight modifications. The crude enzyme obtained during protein extraction in total protein content determination was used to determine PAL activity in the sample. One hundred microliter of crude enzyme obtained were mixed with 1.15 mL containing 0.1 M sodium borate buffer (SBB; pH 8.8) and 10 mM L-phenylalanine. The mixture was then incubated at 37°C for 1 hour following which 250 µL of 5 N HCl was added to stop the reaction. PAL activity was then measured at 290 nm and the result was expressed in nanomoles of trans-cinnamic acid formed per milligram of proteins.
Real-time reverse transcription polymerase chain reaction (RT-qPCR) analysis
The RNA was isolated via RNeasy Plant Mini Kit (Qiagen, Germany) following the protocol described in Lai50. First strand cDNA was converted from 1 μg of isolated total RNA using QuantiNova Reverse Transcription Kit (Qiagen, Germany). Expression profile were assessed via RT-qPCR analysis. Real-time PCR was performed with Bio-Rad CFX96 system (Bio-Rad, US) with QuantiNova SYBR Green PCR (Qiagen, Germany) following the protocol as described in Lai et al.51. The real-time PCR reaction conditions used were as follows: 95 ºC for 30 s followed by 40 cycles of 95 ºC for 5 s and 60 ºC for 5 s. Three technical replicates on three biological replicates were performed on each sample. The data were analyzed using Bio-rad CFX Manager 3.1 software. The relative expression levels (2-ΔΔCT) were calculated according to Livak’s method52. The reference genes used in this study were rice cyclophilin (OsCYC) and ubiquitin 5 (OsUBQ5).
Statistical analysis
All data presented were the average ± standard deviation (SD) of three biological replicates. The Student’s t test was applied in evaluating the level of significant differences at p < 0.05 between the different treatments using the SPSS v.20 software (IBM Corp., Armonk, USA).