Fungal strain and rice seeds
P. liquidambaris was isolated from the inner bark of Bischofia polycarpa (Shi et al. 2004). The fungus was stored at 4°C on potato dextrose agar (PDA, containing 200 g L− 1 potato extract, 20 g L− 1 glucose, and 20 g L− 1 agar, pH 7.0). The rice cultivar was a japonica subspecies of Oryza sativa L. “Wuyunjing 7,” which was a common cultivar grown in the Jiangsu Province of south-eastern China.
Qualitative assay of ACC deaminase activity from P. liquidambaris
ACC was used as a nitrogen source for P. liquidambaris. The activity was measured according to the reported protocol (Siddikee et al. 2010). P. liquidambaris was inoculated into Jensen’s Nitrogen Free medium (JNF) supplemented with ACC (Sigma-Aldrich Co., St Louis, MO, USA). P. liquidambaris couldn’t grow in the medium without nitrogen sources, which indicated the strain had an ability of utilizing ACC.Amplification, cloning and sequencing of ACCD gene from P. liquidambaris.
Protein sequences of ACCD of Aspergillus flavus (XM_002378519.1), Neosartorya fischeri (XP_001265664.1), Penicillium citrinum (AB038511.1), Trichoderma asperellum (FJ751936), Fusarium oxysporum, and Gibberella zeae PH-1 (XP_385209.1) were obtained from the National Center for Biotechnology Information’s (NCBI) database and aligned using the ClustalW program (Thompson et al. 1994). Primer sets generated by iCODEHOP were chosen based on the conserved protein sequences AYGGNK and AFITDPVYEGKS, (Fig. 1 and ST. 1) and obtained from Invitrogen (Springen, Nanjing, China).
Fungus genomic DNA was prepared using AxyPrep Multisource Genomic DNA Miniprep Kit (Axygen Biosciences, Union City, CA, USA) following the manufacturer's instruction. The PCR reaction (50 µL) contained 5 µL 10× PCR buffer, 3 µL 25 mM MgCl2, 1 µL 10 mM dNTP, 1 µL of each primer (25 mM), 2 µL (5.0–10.0 ng) of total DNA, 0.5 U Taq polymerase, and 36.5 µL dd-H2O. DNA were amplified on 96 well thermal cycler (Applied Biosystems, Foster City, CA, USA) and program consisted of an initial denaturation at 95°C for 5 min followed by 30 cycles of 94°C for 60 s, 58°C for 60 s, and 72°C for 60 s with a final extension of 10 min. Samples (10 µL) of the PCR products were separated on a 1% agarose gel in TAE buffer (90 mM L− 1 Tris-acetate, 2 mM l− 1 EDTA, pH 8.3) containing 0.5 µg of ethidium bromide mL− 1 at 120 V for 25 min., visualized under a UV transilluminator and photographed using the gel documentation system (Alpha Innotech Corp). The PCR products were purified using a DNA Gel Extraction Kit (Axygen Bioscience Inc.). Recovered fragments were cloned into the pMD 19-T vector and Escherichia coli DH5a (TaKaRa) competent cells and sent for sequencing to Invitrogen Corp. (Springen, Nanjing, China). Downstream flanking sequences of ACCD gene of P. liquidambaris were obtained by nested-PCR using different nested gene specific primers (ST. 1 and SF. 2) and Universal GenomeWalker Kit (Clontech, Mountain View, CA, USA). The upstream flanking sequence was obtained using the same kit with another set of nested specific primers (ST. 1 and SF. 2). The GenBank accession number of the full sequence of P. liquidambaris ACCD gene is KX762324.
Quantitative assay of ACC deaminase activity
According to the previous reports, the activity of ACCD was analyzed (Siddikee et al 2010). Fungi were cultured in PDA for 72 h at 28°C at 200 rpm, harvested by centrifugation at 6000 ⋅ g for 10 min at 4°C and washed 3 times with sterilized ddH2O. Fungal pellet were transferred to 10 mL JNF medium supplemented with an aliquot of 60 µL of 0.5 M ACC as the sole nitrogen source to induce ACCD activity. Fungi were harvested again by centrifugation and washed by suspending in 5 mL of 0.1 mol L− 1 Tris–HCl, pH 7.0. The pellet was re-suspended in 600 µL 0.1 mol L− 1 Tris–HCl, pH 8.5 and 30 µL toluene and vortexed at the highest setting for 30 s. A 100-µL aliquot of the toluenized mycelia was set aside and stored at 4°C for protein assay at a later time. Two hundred µL toluenized mycelia and 20 µL of 0.5 mol L− 1 ACC were taken in a fresh 1.5 mL microcentrifuge tube and incubated at 30°C for 15 min. Then, 1 mL of 0.56 mol L− 1 HCl were added, vortexed for mixing and centrifuged at 12 000 ⋅ g for 5 min at room temperature. One ml of the supernatant was separated and vortexed together with 800 µL of 0.56 mol L− 1 HCl. Again, 300 µL of the 2, 4-dinitrophenyl hydrazine reagent was added, vortexed and incubated for 30 min at 30°C. Absorbance was measured at 540 nm immediate after adding 2 mL of 2 mol L− 1 NaOH. All sample were carried out in triplicate. ACCD activity was evaluated quantitatively by measuring the amount of α-ketobutyrate produced by the deamination of ACC and expressed as µmol of α-ketobutyrate mg protein− 1 h− 1. Protein concentrations were use the bovine serum albumin (BSA) as standard (Lowry et al 1951).
Ethylene production and growth assay of rice seedling under different levels of salt stress
The rice seeds were shocked in 70% ethanol for 15 min, rinsed twice with sterile water, sterilized for 25 min in 0.1% HgCl2, and rinsed six times in sterile distilled water (Yang et al. 2015). The sterilized seeds and the last washing water were placed on PDA/LB plates and incubated for five days at 28°C as sterility checks (Feng et al. 2006). The sterilized seeds were transferred to petri dishes (20-cm diameter, 100 grains per dish), treated with 80 mL of sterilized deionized water and incubated in a growth cabinet (29°C during the day and 25°C during the night, 16/8 h photoperiod at 250 µmol m− 2 s− 1, relative humidity [RH] = 70%) for 3–4 days for germination. Germinated rice seeds were transplanted into pots (25-cm diameter, 35-cm high) containing sterilized 1.2 kg vermiculite and incubated in a growth chamber in the above-mentioned conditions. Fourteen-day-old seedlings were exposed to different levels of salt stress (100, 150, and 200 mmol L− 1 NaCl) for seven days with 3 split applications to avoid osmotic shock. Ⅰ) Seeds and seedlings not treated with salt were used as negative controls-1 (NC1), Ⅱ) seeds and seedlings treated with AVG (an inhibitor of ACS activity) and salt were used as negative controls-2 (NC2), Ⅲ) seedlings treated with salt were used as positive controls (PC). The experiment was set with three replications.
Based on the previous reports and slight modification, the ethylene emission of rice seedlings was measured (Siddikee et al. 2011ab). Fourteen-day-old rice seedlings were irrigated using sterilized deionized water containing 50, 125, 150, or 200 mmol L− 1 NaCl to create different levels of salt stress and incubated in a growth chamber at 28°C, RH 70%, and light intensity 250 µmol m− 2 s− 1 for 6 h. Seedlings (30 for each treatment) were uprooted and washed using the respective concentrations of saline water to remove soil from the roots and placed inside a 40 ⋅ 7 cm glass tube. The glass tubes were kept open for 30 min to let air escape and then sealed for 8 h using a rubber septum, and the air sample from the headspace was sampled. One-milliliter samples of the headspace were injected into a Gas Chromatograph (Agilent 6850, GC System) packed with a TM-PLOT U column at 70°C and equipped with a flame ionization detector. GC was adjusted at 40°C, 120°C, and 220°C with 50°C min− 2 for oven, injection, and detection temperature, respectively. The carrier gas was He and flow rates of He, H2, and Air were 1.1, 40, and 400 ml min− 1. Make-up flow rate of nitrogen was 25 mL min− 1. The amount of ethylene emission was expressed as pmol L− 1 of ethylene g− 1 dry weight h− 1 by comparing the standard curve generated with pure ethylene. Twenty-one-day-old seedlings (n = 10) from plastic pots for each treatment were carefully removed for morphological and physiological assay. The length and fresh weight of root-shoot were immediately recorded. The root and shoot of 10 seedlings were separated and dried in an oven at 70°C for approximately 3–4 days until a constant weight was obtained and recorded.
The effect of P. liquidambaris inoculation on the ethylene production and growth of rice seedlings under in-vitro salt stress conditions
P. liquidambaris was cultured in 1-L Erlenmeyer flasks containing 500 mL of sterilized PDB for 3 days at 160 rpm in an orbital shaker at 28°C. Ten ml of culture broth was separated to collect mycelia. Approximately 3.0 g (equivalent to ± 0.30 g dry weight) fungal mycelia were washed twice with sterile distilled water, re-suspended in JNF medium supplemented with 0.5 mmol L− 1 ACC as sole nitrogen source and incubated for 24 h at 30°C at 200 rpm to induce ACCD activity. Mycelia were harvested, washed, and re-suspended in sterile distilled water and diluted to a final volume of 200 mL. This suspension was used as the fungal agent for the germinating seeds to facilitate colonization. The thoroughly sterilized (see previous section) seeds were randomly divided into two groups and transferred to petri dishes. For the inoculated group (E+), 80 mL of the above-described fungal agent and for the non-inoculated group (E−) 80 mL of sterilized deionized water was added to each dish (Tian et al. 2007). Seeds were germinated and grown for 3–4 days in growth cabinet, transplanted into pots and further grown in growth cabinet in the conditions already mentioned above. Rice seedlings were subjected to the following six treatments; 1) Negative control (NC1): no-inoculation and no salt; 2) NC2: no inoculation, 0.03 mol L− 1 MgSO4 salt and AVG; 3) Positive control (PC): 0.03 mol L− 1 MgSO4; 4) PC + P. liquidambaris inoculation (PS + E+): 0.03 M MgSO4 + inoculation; 5) 150 m mol L− 1 NaCl stress (SS): seedlings exposed to 150 m mol L− 1 NaCl stress; and 6) SS + P. liquidambaris inoculation (SS + E+): seedlings inoculated with fungi and exposed to 150 m mol L− 1 NaCl stress. Salt stress was applied to 14-day-old seedlings in 3 split application to avoid osmotic shock. Fertilization was performed in the form of irrigation of Hoagland nutrient solution as necessary. Three biological replicates per treatment were established. Ethylene production from rice seedling was assayed as described above. Data related to growth of rice seedlings were recorded from 21-day-old seedlings as described above. Based on the data obtained, the salt tolerance index (STI) of rice seedlings was determined (Siddikee et al. 2011a) as: STI = DWS or DWH/DWC (where, DWS = dry weight of plant grown under salt stress, DWH = dry weight of plant grown under salt stress with inoculation of P. liquidambaris and DWC = dry weight of plant grown in control condition (without salt stress and inoculation of P. liquidambaris). Meanwhile, 18–20 seedlings from each treatment were stored at -80°C and subsequently used for physiological assay such as ACC content, ACS and ACO activity, chlorophyll content of the leaves and ACCD activity.
Assessment of the effect of rice seedlings inoculated with P. liquidambaris on ACC content and ethylene biosynthesis-related enzyme activities
Based on the existing methods, some modifications were made to measure the ACC concentration in the stems of rice seedlings (Siddikee et al. 2011b). One gram of shoot sample was immediately frozen in liquid nitrogen and ground using a mortar and pestle. ACC from frozen ground tissue was extracted in 5 mL 80% methanol containing butylated hydroxytoluene (BHT, 2 mg L− 1) as an antioxidant and incubated at room temperature for 45 min. Samples were centrifuged at 2,000 ⋅ g at 20°C for 15 min and were re-suspended in 4 mL methanol and again centrifuged. The combined supernatants were evaporated to dryness under a vacuum in a rotatory evaporator. ACC was determined according to the existing detection scheme (Wachter et al. 1999). Residues were re-suspended in 2 mL distilled water and the upper aqueous phase (0.5 mL) obtained by extraction with dichloromethane was mixed with 0.1 mL HgCl2 (80 mM) in test tubes and sealed with rubber septa. Then, 0.2 mL NaCl solution (40 ml NaOH, 80 mL 12.5% NaCl solution, and 30 mL distilled water) was injected into the tubes, shaken, and incubated for 8 min. One milliliter of the gaseous portion was removed and assayed for ethylene by gas chromatography (GC).
Determination of ACC synthase (ACS) and ACC oxidase (ACO) activities for rice seedlings inoculated with P. liquidambaris
ACS and ACO was the key enzyme for ethylene biosynthesis. The enzyme extracts used to measure the activity of ACS and ACO in vitro were modified according to the existing reports (Siddikee et al. 2011b). One gram shoot sample was homogenized by pulverization with a mortar and pestle in liquid nitrogen. Then, 4 mL of 100 mol L− 1 Na-phosphate (pH 9.0) containing 5 µmol L− 1 pyridoxal phosphate (PLP), 4 m mol L− 1 2-mercaptoethanol (2-ME), 1 m mol L− 1 EDTA, and 10% glycerol were added in the presence of 1 g polyvinylpolypyrrolidone (PVPP). Ammonium sulfate (35 and 75% saturation) was added to the enzyme extract to induce precipitation and the precipitate was re-suspended in 2.5 mL of a solution containing 100 m mol L− 1 Na-phosphate (pH 7.8), 5 µmol L− 1 PLP, 0.5 m mol L− 1 2-ME, and 10% glycerol. ACS activity was assayed by incubating a 100-µL enzyme solution with 100 m mol L− 1 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES)–KOH (pH 8.5), 5 µM PLP, 100 µmol L− 1 S-adenosyl-L-methionine (SAM), and test chemicals at given concentrations in a total volume of 400 µL in test tubes and sealed with rubber septa. The reaction mixture was incubated for 15 min at 30°C and the amount of ACC was determined as described above.
For assaying the ACO activity, frozen tissues were pulverized in liquid nitrogen and homogenized in 2 mL g− 1 of extraction buffer consisting of 100 m mol L− 1 Tris–HCl (pH 7.2), 10% (w/v) glycerol, and 30 m mol L− 1 sodium ascorbate. The homogenate was centrifuged at 25,000 ⋅ g for 15 min at 4°C. The supernatant obtained was used for the in vitro ACO assay (Siddikee et al. 2011b). Enzyme activity was assayed at 30°C for 15 min in 10-mL screw-cap tubes fitted with a Teflon-coated septum containing 1.5 mL of supernatant, 50 µmol L− 1 FeSO4, 1 m mol L− 1 ACC, and 5% (v/v) CO2. After the incubation period, the quantity of ethylene released into the headspace was determined by GC.
AAnalysis of ACCD gene from P. liquidambaris by qRT-PCR
Approximately 0.1 g of mycelia (wet weight) was harvested and washed using ddH2O. Mycelia were frozen in liquid nitrogen, ground to powder using mortar and pestle and total RNA was extracted using an E.Z.N.A fungal RNA Kit (Omega, Bio-Tek, Winooski, VT, USA). RNA concentration was measured at 260 and 280 nm using NanoDrop™ 8000 Spectrophotometer and adjusted. Total RNA was used as the template to synthesize the first strand complementary DNA (cDNA) using a PrimeScript RT Reagent Kit with gDNA Eraser (TAKARA, Tokyo, Japan). The product was characterized by electrophoresis on 1.5% agarose gels and 2 µL product used as a template for qRT-PCR. qRT-PCR was performed on the StepOne Real-time PCR system (Applied Biosystems), using SYBR Premix Ex Taq Kit (Tli RNaseH Plus) (TAKARA). The specific primer pairs used for quantitative measurement of the transcription of ACCD gene were designed according to their partial mRNA sequence (ST. 1 and SF. 2). Each 20 µL qRT-PCR reactions mixture contained 2⋅ SYBR Premix Ex Taq (TAKARA), 10.0 µM each gene-specific primer, 0.4 µL of 50⋅ ROX Reference Dye, and 2.0 µL of cDNA template. The qRT-PCR cycling conditions were 95°C for 1 min, followed by 40 cycles of 95°C for 15 s, 60°C for 45 s, and 72°C for 30 s. The specificity of amplification was verified by a standard melting curve. The primers used for amplification of the ITS gene of P. liquidambaris used as an internal control are presented in ST. 1. The relative abundance of mRNA was normalized against the levels of ITS Amplification data were analyzed using the delta-delta-Ct (ΔΔCT) method (Livak and Schmittgen, 2001). Each sample was amplified in triplicate in each experiment.
Analysis of ACO, ACS, and ACCD gene expression from plant tissues by qRT-PCR
Total RNA was extracted from the shoots of 15-day-old rice seedling using a Plant RNase Mini Kit (Qiagen, Hilden, Germany) and subjected to RT-PCR. RNA concentration assay, cDNA synthesisand qRT-PCR were performed as described above. The specific primer pairs used for quantitative measurement of the transcription of ACO and ACS genes were designed according to their conserved mRNA sequence in rice as follows: ACO (accession: LOC_Os04g10350), ACS (accession: M96672.1) and ACCD gene (accession: LOC_Os02g53330 and LOC_Os01g50060); and actin (Yang et al. 2012) was used as an internal control (ST. 1). The relative abundance of mRNA was normalized against the levels of actin.
Bioinformatics and statistical analysis
The nucleotide sequences of the ACCD gene were translated into amino acid sequences using http://www.fr33.net/teranslator.php and http://www.expasy.org. Molecular weight and isoelectric point of the proteins were predicted by using http://www.expasy.org. The conserved domains were predicted and analyzed by using the conserved domain database (CDD) (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Phylogenetic trees of nucleotide sequences and amino acid sequences of the ACCD gene were constructed using the MEGA7.0. Statistical analysis was performed using SPSS software (version 18.0, Chicago, IL, USA). All graphs were generated using Origin software version 8.0. All data are reported as the average of three biological replicates ± standard deviation (SD). One-way ANOVA and LSD test were used to determine significant differences in means among treatments.