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L. Chinense Growth Conditions and Artificial Pollination

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The genus Liriodendron is a distinctive and valuable hardwood that has great ecological and economic values. It grows fast and the wood is light and soft, so it is cultivated in many temperate mountains of the world for wood production [1-4] and it’s recommended for waste landfill remediation [5]. It's a flowering plant with beautiful leaves and hence used for urban landscaping as it provides shading as well. Besides, the tree genus Liriodendron is valued as materials source for honey production, chemical extracts [6-8], biomass and biofuels [9, 10].

The genus Liriodendron survived from the last Ice Age and was distributed in large geographical ranges of North American and East Asian respectively. Currently it comprises only two morphologically similar species, Liriodendron tulipifera L. and Liriodendron chinense (Hemsl.) Sarg. [11]. However L. chinense (Liriodendron chinense (Hemsl.) Sarg.) has been regarded as a rare and endangered plant because it occurs in small, isolated and thinly scattered populations [12]. L. chinense was listed in the IUCN Red List of Endangered Plants in China [13], and has currently been classified as a lower risk or near-threatened species (http://www.iucnredlist.org/).

In the process of L. chinense sexual reproduction, the low seed setting percentage is a marked trait. After years of statistics, the setting percentage of L. chinense is not more than 10% in natural state, and it is hard to find the seedling in natural environment [14]. In the last two decades, many researchers have conducted studies, such as examining the relative contribution of the pollen fertility and transfer, availability of resources, flower or seed predation and genetics, to find out why L. chinense only produce few seeds [15-18]. Unfortunately, there has been no consistent conclusion. Pollination, as a key event in reproductive process of plants, especially in rare or endangered plant species like L. chinense that have low seed production, is probably one of the weak links in the reproductive cycle. Any barrier occurring between pollen and stigma interaction will lead to low seed production, however, few studies have focused on the pollination in L. chinense. Zhou and Fan examined the pollen quality, pollen germination and growth on stigma using fluorochroma method. The results indicated that in vivo the pollen grains can load on about 64% pistils of the gynoecium, but the rate of pollen tube passing the style is low, only 24% [19]. In addition to few pollen tubes passing the style, the pollen tubes may grow twined or in no direction, suggesting that only a smaller percent of the pollen tubes penetrates the micropyle and enter into ovule [20, 21]. These results show that the interaction between pollen and stigma occurs in different phases after pollen grains loading on stigma, and there are different barriers distributed in stigma surface, style and ovule during pollen tube growth.

In self-compatible plants, the pollen-stigma interaction comprises six stages between pollen and pistil: pollen capture and adhesion, pollen hydration, pollen germination, penetration, growth of pollen tube through the stigma and style, pollen tube enter into the ovule and discharge the sperm cells [22]. After the pollen-stigma interaction, the nuclei of two gametes fuse to form the zygote. However, in self-incompatible plants, no matter the barriers occurs in which stage of interaction, there is no formation of a viable zygote. Previous studies in L. chinense showed that many pollen grains germinated on pistils of the gynoecium but few pollen tubes could penetrate the pistil style, and most of the pollen tube couldn’t pass through micropyle and enter into ovule. This phenomenon suggests that there might be other factors affecting pollen-stigma interaction in L. chinense. To verify this hypothesis, we conducted a systematic morphological and proteomic analysis on the pistil of L. chinense during pollination. The result provides new insights in the mechanism underlying sexual reproduction in L. chinense.


L. chinense growth conditions and artificial pollination

The L. chinense plants was grown in Wuhan Botanical Garden, Chinese Academy of Sciences. During the flowering season, which extends from late April to May, the branches with flower buds which were about to open were cut from the tree and cultivated with half-strength Hoagland’s nutrient solution in greenhouse under 14 h light (400-800μmolm-2s-1) at 26±2oC and 10 h darkness at 20±2 oC [23]. The relative humidity was maintained at 60-70% [19]. The flower buds with an opening on top and a probability of opening the following day were chosen and the androecium was emasculated at night before pollination. Artificial pollination was done the next afternoon as follow: Mature pollen grains were harvested from open flowers and then were smeared on the pistils without androecium using a soft brush. This artificially pollinated pistil was cut from the flower 30 minutes after pollination and stored in liquid nitrogen. Similarly, the pistil after 1 h pollination was harvested, stored in liquid nitrogen. The harvested un-pollinated pistil was stored in liquid nitrogen. All three of these samples were named as S2, S3, and S1 respectively and stored in -80 oC freezer. All three treatments (S1, S2, and S3) were repeated five times respectively.

Paraffin section

Anthers and pistils were fixed in FAA solution containing 5% glacial acetic acid, 5% formaldehyde, 70% ethanol at room temperature for 24 h. After dehydration and infiltration, the samples were embedded in paraffin and cut into 10-µm-thick sections by Rotary Microtome Leica RM2265 (Germany). Then the sections were sealed by neutral balsam and photographed by Olympus-BX51 (Japan).

Gel-based proteomics in L. chinense

Protein extraction and 2-DE

Proteins of pistils were extracted as previously described [24]. Briefly, 0.25-0.3 g of pistils were ground in 2 ml pre-cooled homogenization buffer which contains 20 mM Tris-HCl (pH7.5), 250 mM sucrose, 10mM EGTA, 1% Triton X-100, 1 mM PMSF, and 1 mM DTT. The homogenate was shifted into a centrifugal tube and centrifuged at 12000×g for 30 min at 4 oC. The supernatant was collected in new centrifugal tube and mixed with 3 volumes cold acetone. The tube was kept at -20 oC at least 2 h, and then centrifuged at 12000×g for 30 min at 4 oC, and the precipitate was collected and washed with cold acetone three times. After centrifugation, the pellet was vacuum-dried. The immobilized pH gradient strips (17 cm, pH 4-7 linear, Bio-Rad, USA) were loaded with 350 μl sample buffer containing 800 μg sample proteins at room temperature in tray for 16 h. Isoelectric focusing was performed with the PROTEAN IEF system (Bio-Rad, USA) for a total 80000 V-hr. Then the strips were equilibrated in equilibration buffer I (6 M urea, 2% SDS, 0.375 M Tris-HCl pH 8.8, 20% glycerol, and 130 mM dithiothreitol) for 15 min and equilibration buffer II (6 M urea, 2% SDS, 0.375 M Tris-HCl pH 8.8, 20% glycerol, and 135 mM iodoacetamide) for 15 min sequentially. After equilibration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out with 12% acrylamide gels. The 2-DE gels were stained with Coomassie Brilliant Blue (CBB) R-250.

Image analysis of 2-DE gels

The 2-DE gels were scanned at 600 DPI resolutions with an EPSON PERFECTIONTM V700 PHOTO scanner (Epson (china) Co., Ltd.). The images were analyzed with PDQuestTM 2-DE Analysis Software (Version 8.0, Bio-Rad, USA). Spot volumes were normalized by total spot volumes per gel to avoid experimental variations among 2-DE gels. Comparisons and statistical analysis were performed using the calculated average values of each biological replicate among the three different treatments. The protein spots with more than a two fold change among treatments and that passed a Student’s t-test (P < 0.05) were considered to be differentially expressed spots.

Protein identification by MALDI-TOF/TOF-MS

The significant differentially expressed spots were excised from the gel manually, and washed with double distilled water twice for 20 min, then distained with 100 μl of 50 mM NH4HCO3 in 50% v/v acetonitrile (ACN) for 1 h until the gel is mostly colorless at room temperature. The liquid was removed and 50 μl ACN was added to dehydrate the gel. After drying the gel, 25 mM NH4HCO3 containing 10 pmol trypsin (Promega, Madison, WI, USA) was added to the tube and kept at 4 oC for 1 h, and then it was kept at 37 oC overnight. The proteins were then digested according to the method described before [25]. The peptides were extracted and collected using three kinds of solution (0.1% TFA/99.9% acetonitrile, 0.1% TFA/99.9% H2O, 0.1% TFA/50% acetonitrile/49.9% H2O) from gel spot. The peptide solution was concentrated to 10 μl, and then desalted by ZipTip C18™ pipette tips (Millipore, Bedford, MA, USA). After trypsin digestion, the protein peptides were dried by SpeedVac. Then peptides were dissolved in 0.1% trifluoroacetic acid, and then 1 μl of the sample solution was loaded on Anchor Chip Standard (Bruker Daltonics Inc, Germany). After the Anchor Chip drying, the matrix solution (20 g/L HCCA, TA 95%) was loaded on point corresponding to the location of the sample to a target spot. Through ultrafleXtreme (Bruker Daltonics Inc, Germany) Operation, the PMF data was obtained. The instrument parameters for MS acquisition were list as follows: laser intensity was 20%-26%, reflector detector voltage was 2438 V. Protein identification using MS/MS raw data was performed with flexAnalysis software (Bruker Daltonics Inc, Germany) coupled with Mascot Server software (version 2.4.01) based on the NCBI protein database and SwissPort database of green plants. The searching parameters were set as follows: peptide masses were assumed to be monoisotopic, 100 ppm was used as mass accuracy, a maximum of one missing cleavage site, and modifications which included Carbamidomethy and Oxidation were considered. (The timestamp of NCBI protein database is 2011/11/09, there were 949,856 sequences of Green Plants and 5,512,397,590 redundant total sequences in NCBI database; the timestamp of SwissPort 57.15, there were 28,783 sequences of Green Plants and 515,203 sequences non-redundant total sequences in SwissPort). The proteins which scores greater than 42 (NCBI) or 26 (SwissPort) (P<0.05) were chosen. Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits.

Gel-free proteomics in L. chinense

Protein extraction

The protein samples for iTRAQ were recovered in lysis buffer (30 mM Tris-HCl, pH 8.5, 7 M urea, 2 M thiourea, and 4% [w/v] CHAPS) by phenol extraction and methanol/ammonium acetate precipitation as described previously [26]. The protein pellets were resuspended in buffer (7 M urea, 2 M thiourea, 4 % CHAPS and 10 mM DTT) in a minimal volume and protein was quantified using BCA protein assay kit (Pierce, USA).

Digestion and iTRAQ labeling

About 100 μg proteins of each sample per tube were prepared. Then it was reduced by adding DTT to a final concentration of 12 mM and incubated for 1 h at 37 oC. Subsequently, iodoacetamide was added to a final concentration of 50 mM, and the mixture was incubated for 1 h at room temperature in the dark. Then the mixture was transferred to centrifugal units (VN01H02, Sartorius, Germany) and centrifuged at 12,000×g for 20 min, and then the filtrate was discarded. Subsequently, 8 mM urea solution was added into the centrifugal units and centrifuged, repeated this step twice. After that, 100 μl dilute buffer (50 mM triethylammonium bicarbonate) was added into the centrifugal units and centrifuged. Then 50 μl dilute buffer containing 2 μg modified trypsin (Promega) was added into the centrifugal units at 37 oC overnight. The resulting peptides were then labeled with iTRAQ reagents (AB Sciex, USA) according to the manufacturer’s instructions. For each time point (i.e., S1, S2, and S3), each sample was iTRAQ labeled 3 times except S3. (i.e., 113-, 116-, 119-iTRAQ tags for S1 3 replicates. 114-, 117-, 121-iTRAQ tags for S2 3 replicates. 115-, 118- iTRAQ tags for S3 2 replicates.)

MS/MS Analysis

Then the mixture of labeled peptides was concentrated and acidified to a total volume of 2 mL. Labeled peptides were desalted with C18-solid phase extraction and dissolved in strong cation exchange (SCX) solvent A (25% (v/v) acetonitrile, 10 mM ammonium formate, and 0.1% (v/v) formic acid (pH 2.8). The peptides were fractionated using an Agilent HPLC system 1260 with a polysulfoethylA column (2.1 × 100 mm, 5 µm, 300 Å; PolyLC, Columbia, MD, USA). Peptides were eluted with a linear gradient of 0–20% solvent B (25% (v/v) acetonitrile and 500 mM ammonium formate (pH 6.8) over 50 min followed by ramping up to 100% solvent B in 5 min. The absorbance at 280 nm was monitored, and a total of 37 fractions were collected. The fractions were combined into 12 final fractions and lyophilized. A quadrupole time-of-flight (LTQ Orbitrap XL) MS system (Thermo Fisher Scientific, Bremen, Germany) was applied as described previously [27]. It interfaced with an Eksigentnano-LC AS2 system (Eksigent Technologies, LLC, Dublin, CA) using high energy collision dissociation (HCD). Each fraction was loaded onto an Agilent Zorbax 300SB-C18 trap column (0.3 mm id × 5 mm length, 5 µm particle size) with a flow rate of 5 µl/min for 10 min. Reversed-phase C18chromatographic separation of peptides was carried out on a pre-packed BetaBasic C18PicoFrit column (75 µm id × 10 cm length, New Objective, Woburn, MA) at 300 nl/min using the following gradient: 5% B for 1 min as an equilibration status; 60% B for 99 min as a gradient; 90% B for 5 min as a washing status; 5% B for 10 min as an equilibration status (solvent A: 0.1% formic acid in 97% water, 3% ACN; solvent B: 0.1% formic acid in 97% ACN, 3% water).

Database Search and Quantification

The MS/MS data were processed by a thorough search considering biological modification and amino acid substitution against non-redundant NCBI green plants 20131014.fasta (1,544,439 contigs) under the Sequest®algorithm of Proteome Discoverer.1.4 software (Thermo Fisher Scientific Inc.). Protein function analysis by blast2go software (http://www.blast2go.com/b2ghome) was conducted according to the early literature [28-31].

The search results were passed through additional filters before exporting the data. For protein identification, the filters were set as follows: significance threshold P<0.05 (with 95% confidence) and ion score or expected cutoff < 0.05 (with 95% confidence). For protein quantitation, the filters were set as follows: only unique peptides were used to quantify proteins. “Weighted” was chosen for protein ratio type (http://mascot-pc/mascot/help/quant_config_help.html); S2/S1 or S3/S1 ratios >2 and <0.5 are considered significantly up- and down-regulated.

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