Plant material
Young leaf and internodes of Bambusa balcooa (Roxb.) were collected from natural strands of Bhadreswar (22° 49′ N 88° 21′ E), West-Bengal, India.
RNA ligase mediated rapid amplification of cDNA ends (RLM RACE)
Total RNA was isolated from the fifth internodal tissue of bamboo following protocol adapted by Rai et al. [32]; RACE was performed using First Choice RLM RACE kit (Invitrogen, USA) following manufacturer’s instructions. Primers used in 5′ RACE were designed using the information of 5′ end sequence of the marker, Balco1128. Similarly 3′ end sequence information was used to design primers for 3′ RACE (Additional file 8: Table S2). Products obtained from RACE were gel purified using Qiagen gel extraction kit (Cat No 28704; QIAGEN; USA) and sequenced.
Genomic DNA isolation and genome walking
The PCR based genome walking library was constructed by Genome Walker Universal Kit (Cat no. 638904; Clonetech, U.S.A). Briefly, high quality genomic DNA was isolated from surface sterilized tissue with 250 μl of warm CTAB extraction buffer [33], and digested with four different restriction enzymes namely, DraI, EcoRV, PvuII, StuI for overnight at 37°C. The digested DNA was purified, and ligated with Blunt end adaptors (25 μM, provided with the kit) for overnight at 16°C. Adaptor ligated DNA was used as template for first round genome walking PCR employing both adaptor specific (AP1, provided with the kit) and gene specific primers (Additional file 9: Table S3). The longest product was eluted from the 1% agarose gel and sequenced with BigDye Terminator Reaction Mix (Applied Biosystems, USA) using an automated DNA sequencer (ABI Prism 3100) following manufacturer’s instruction. Four rounds of walking were required to obtain the full length BbKst clone.
In-silicosequence analysis
Both the sequences obtained from genome walking and RACE were aligned by NCBI blast alignment using Bioedit software. Positions of promoter sequence, transcription start site, introns and exons were determined by NCBI- blast and by multiple alignments with ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The in silico translated amino acid sequence derived from the BbKst gene was further analyzed for conserved domains and motifs using ProSite and Inter-Proscan.
Bacterial expression of BbKst- kinase cDNA
Expression cassette construction
The kinase domain of BbKst (from 2233 to 3342 bp of cDNA as shown in Figure 1C) was cloned in EcoRI and SalI sites of pET28a(+) vector. Subsequently transferred in BL21 cells, plasmid DNA obtained from a positive clone was sequenced with gene specific primers for sequence conformation. BbKst-kinase-His fused protein was induced with IPTG and purified using Ni- NTA nickel column (Cat no. 31014; Qiagen) following manufacturer’s instruction.
In vitrokinase assays
Substrate phosphorylation experiments were carried out employing 10 μg of BbKst-kinase in 50 μl of reaction mixture (25 mM Tris-HCl; pH 7.5, 1 mM DTT; 5 mM MgCl2 and 10 μM ATP) in presence of 10 μM [γ -32P] ATP (3000 cpm pmol-1) at 30ºC with 250 mM of different substrates viz. casein, myelin basic protein (MBP), histone (H1), LRRKtide and GS peptide. Phosphorylated products were analysed by SDS-PAGE and autoradiographed. Activity of BbKst-kinase was determined by plotting its specific activities with varying concentrations of casein as substrate. Steady-state kinetic parameters were evaluated using Michaelis-Menten kinetics. The V
max
and Km value of BbKst-kinase using casein as a substrate following Eadie-Hofster plots of V0 versus V0/[S]. The turnover number was calculated from Kcat at the optimal substrate concentration per active site and the average time required to hydrolyze one molecule of casein was determined.
Effect of protein kinase inhibitors of BbKst-kinase activity
Several potent inhibitors of protein kinases like heparin, H-89, quercetin and staurosporine were tested on BbKst-kinase following recommended methods of Davies et al. [34].
Phospho-amino acid analysis
The purified BbKst protein was labeled in vitro with [γ-32P] ATP and autophosphorylated protein electroblotted onto a polyvinylidine difluoride membrane. Radioactive band of interest was excised and hydrolyzed in 6 N HCl for 2 hr at 110°C [35]. The hydrolysate was concentrated and spotted on TLC plate and analysed along with Phosphoamino acid standards such as phospho- Ser, phospho-Thr, and phospho-Tyr (25 nM each; Sigma). TLC plates was then exposed to X-ray film and subsequently developed, phosphoamino acids standards were visualized by ninhydrin spraying over the TLC plate.
RNA slot blot hybridization
Different intermodal tissues (viz. I2, I5, I8, I10, I12, I15) were collected from three independent clumps of B. balcooa separately and processed for extracting total RNA as described earlier (35). Twenty μg RNA of each sample were loaded in formaldehyde denatured agarose gel and electrophoresed at a constant voltage of 50 V. The RNA was transferred to Hybond-N + membrane (Amersham-Pharmacia Biotech) with Bio-Rad Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell at a constant current of 400 mA for 45 min. 1265 bp long cDNA fragment was radiolabelled with [α-32P]dCTP using Prime-a-gene labeling system kit (Promega, USA) as per the manufacturer’s instructions. The membrane was incubated with the 32P labeled gene specific probe at 65°C overnight in hybridization buffer (Sigma). After hybridization the membrane was washed twice with 2× SSC containing 0.1% SDS for 30 min each at 65°C followed by washing for 30 min (2 times) in 1× SSC and 0.5× SSC containing 0.1% SDS at 65°C. The membrane was exposed to X-Ray films for 24 hr. with intensifying screen and films were developed. The same procedure was followed for GAPDH as control. All experiments were repeated three times.
qPCR analyses of BbKsttranscripts in bamboo
cDNA was synthesized from the total RNA of leaf, stem, different internodal tissues (2nd, 5th, 8th, 10th, 12th and 15th from the tip) and rhizome of B. balcooa using First strand cDNA synthesis Kit (Cat # K1622. Fermentas). qPCR reactions were performed in presence of gene specific primers using a LightCycler PCR System qPCR instrument (MJ Research, Bio-Rad; Model CFD-3220). Fold differences in the BbKst transcript level were quantified using the 2ΔΔCT method [36, 37], considering fold difference of GAPDH as 1.0.
Construction of plant expression vector and Agrobacteriamediated transformation
The full length 3339 bp cDNA of BbKst was inserted between Spe1 and Nco1 restriction sites in a sense orientation in pCambia 1304. Positive construct was designated as Js1304 (Figure 9). Agrobacterium tumefaciens strain C58C1 was transformed with Js1304 by heat shock method. Js1304 integrated-Agrobacteria mediated transformation of tobacco was performed following the protocol of Kumar et al. [38]. Ten independent transgenic tobacco lines were generated with the Js1304 construct and maintained in the green house until seeds were mature. Seeds were harvested and germinated in presence of 30 ug-ml hygromycin and segregation ratio was analyzed.
Transient expression of the BbKst
Gold-spermidine mixture (25 mg of 1 μm gold particles + 100 μl spermidine) was added to 25 μg of Js1304 plasmid DNA and mixed prior to the addition of 100 μl of 1 M CaCl2. The onion peels were placed on the wet filter paper and kept in a Petridish. The gold particles were fired at 200 psi from a 3 cm distance of a Particle Gun (Helios Gene Gun, Bio-Rad make). Each onion peel was put in the Petri dish keeping the epidermal side in contact with the Murashige and Skoog’s (MS) medium [39]. Petridishes were sealed with parafilm and kept at 23˚C for 24 hr. Each onion piece was taken on a glass slide and GFP fluorescence was visualized and image captured in confocal laser scanning microscope (CLSM) using LAS AF (Leica Application Suite Advanced Fluorescence, 1.8.1) software with a confocal pinhole set at “Airy” 5.50 and zoomed to a factor of 1.4× for improved 8- bit resolution. Materials were excited at 488 nm wave length and emission was at 518 nm.
GUS histochemical assay
Histochemical GUS assay was performed following the method of Jefferson [40] in order to monitor the transient gene expression in 21 days old T1 plants. Tissues were incubated in 1 mM chromogenic substrate XGlcA (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid) containing GUS substrate solution for overnight at 37°C. Then tissues were fixed in the fixative for at least 4 hrs followed by treatment with 50% and 100% ethyl alcohol for complete decolorization. Finally tissues were transferred to GUS fixative solution and examined under light microscope and photographed.
Southern hybridization with partial kinase domain of BbKstas probe
Total genomic DNA from young leaves of GUS-positive transgenic plants and vector-transformed plants was isolated following the protocol mentioned above. For Southern analysis, approximately 10 μg of genomic DNA was digested with 100 units (about 10 U/μl) of two enzymes Spe1 and Nco1, electrophoresed in a 0.8% agarose gel and subsequently transferred to a Hybond-N + membrane (Amersham-Pharmacia Biotech). DNA fragment of 1265 bp was amplified with JNWF1 (5′-CTAAAAGGCACCCTGATATGGACAGCATC-3′; Additional file 9: Table S3); and JF3R (5′-GCAAGAAGCATGTACCTATTGAC-3′) primers and then eluted from the gel, 25-50 ng of the eluted DNA was used as a probe after labelling with [α-32P]-dCTP using Prime-a-gene labeling kit (Promega, USA) as per manufacturer’s instructions. Hybridization was carried out overnight at 65°C. After hybridization the membrane was washed twice with 2× SSC buffer containing 0.1% SDS for 30 min each at 65°C followed by washing for 30 min (2 times) in 1× SSC and 0.5× SSC buffers containing 0.1% SDS at 65°C. The membrane was covered with the saran-wrap, exposed to X-ray film (Kodak) with intensifying screen and kept at -80 ºC for 48 hr.
Expression analysis of BbKstin transgenic tobacco plant
Expression analyses of BbKst in transgenic tobacco plants and empty vector transformed plants were carried out in Light-Cycler PCR System qPCR instrument (MJ Research, Bio-Rad; Model CFD-3220). Primers used for qPCR were 5′-CCTCGGTGTTCCTTCCCTTCTGTC-3′ and 5′-GTCAATAGGTACATGCTTCTT GC-3′. The reaction conditions were as follows: initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 94°C for 45 sec, annealing at 56°C for 30 sec, and extension at 72°C for 1 min, final elongation step at 72°C for 10 min. Melting curve was analysed by continuous monitoring of fluorescence between 60°C and 95°C with 0.5°C increment after every 30 sec. Tobacco 18S rRNA house keeping gene was used as internal control. Fold differences in the BbKst transcript level were quantified using the 2-ΔΔCT method [36, 37], considering fold difference of 18S rRNA as 1.0.
Measurement of stem thickness of transgenic tobacco plants expressing BbKstgene
The stem thickness (diameter) of 5 month old transgenic tobacco plants and empty vector-transformed control tobacco plants grown in green house conditions (28°C and 78% humidity) were measured with slide calipers. Measurements of 7th to 20th internodes from tip of these plants were taken. Three independent measurements from each internode were noted and the mean value was scored. Width of the xylem zone (xylem vessels and xylary fibers) of independent transgenic and control plants were measured under the microscope from at least 5 samples and the mean value was tabulated. Width of xylem zone (containing xylary fibers and vessels) of five independent transgenic lines was compared with respective stem width. Similar comparison was also made for one vector-transformed tobacco plant.
Physical characteristics of fiber cells
Small slivers were prepared from 19th internode (from the tip) by macerating the tissue with 67% nitric acid, boiled in a water bath for 10 min and subsequently washed thoroughly in distilled water following the protocol of Bhattacharya et al. [26]. Measurements of fiber diameter, cell wall thickness and lumen diameter (i.e. fiber diameter - cell wall thickness) of 25 intact fiber cells isolated from five T1 transgenic plants were taken. The following equations were used to derive the physical traits: slenderness ratio = fiber length/fiber diameter, flexibility coefficient = (fiber lumen diameter/ fiber diameter) X 100 and Runkel ratio = (2 X fiber cell wall thickness)/lumen diameter [26]. Duncan’s Multiple Range Test (DMRT) was performed to analyze the results proximity with each other, the test was made in SPSS statistical software.
Histological analysis of cellulose contents of fiber cells
Transverse hand sections were taken from the 18th internodes (from tip of the stem) of vector- transformed and transgenic tobacco plants. Sections were stained with 0.2% aqueous solution of Congo Red (CR; Sigma, C6767) overnight in the dark following the standardized protocol prescribed by Bhattacharya et al. [26]. Briefly, the stained sections were washed with deionized water, and images were acquired using CLSM with a confocal pinhole set at ‘Airy’ 1.0. A 30% Argon laser with Acousto-Optical Tunable Filter (AOTF) for 514 nm (20 mW) was used for exciting CR-labeled samples, and red fluorescence emissions were acquired between 560 and 800 nm with the PMT detector gain set at 1100 V. Fluorescence was recorded after passage through a triple dichroic filter 458/514/594. Images were captured using a HCX PL APO objective (63X/NA 1.40) under the above stated conditions.
Estimation of cellulose contents in transgenic plants
The α-cellulose content was estimated following the colorimetric method based on anthrone reagent [26, 41]. Approximately 1.0 g of 3rd internodal tissues from base were crushed, mixed with 3 ml of nitric acid and acetic acid solution (1:8 v/v) and refluxed in boiling water for 30 min. Lignin, hemicellulose and xylosans were removed through successive three washing followed by centrifugation at 10000 rpm for 10 min. The resultant pellet was dissolved in 67% sulphuric acid (v/v), mixed with chilled anthrone reagent (HiMedia Laboratories, India), and incubated for 20 min in a boiling water bath. Cellulose estimation was made spectrophotometrically at 620 nm (Beckman-Coulter, DU-520). The α-cellulose content was estimated from the standard curve prepared using different known concentrations of cellulose (Merck, Germany) and was expressed as the gram percentage of fiber fresh weight.
