Characterization Of Sclereid Structure And Composition And Coloning Of Sclereid Related Enzyme Genes In Pear (Pyrus) Fruit

Sclereid or stone cell is one of the crucial factors impacting grit texture in pear fruit. It is reported that genetic background is the intrinsic factor for sclereids development; however the culture practices can also influence their development consumingly. Unfortunately, no great progress is achieved on pear sclereids research till now; researchers always omit lignin-the chemical nature of sclereids, and few papers are found focusing on pear sclereid lignin directly. In this study,’Dangshansuli’pear which contains high sclereid content was used as the major material, and’Kousui’which has low content of sclereid as accessorial material to characterize sclereid structure and composition; also, PAL (Phenylalanine Ammonia Lyase), CAD (Cinnamyl Alcohol Dehydrogenase) and POD (Peroxidase) genes were cloned and analysed. The results are as follows:1. Anatomy, ultrastructure and lignin distribution in stone cells from the fruit of two Pyrus species (Pyrus bretschneideri cv. Dangshansuli and Pyrus pyrifolia cv. Kousui) were examined by using light microscopy (LM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray energy spectrometer and inductive coupled plasma emission spectrometer (ICP) as well as autofluorescence analysis. Sections stained with phloroglucinol-HCl revealed the presence of lignin in stone cells, and showed that stone cells were distributed in a mosaic pattern in the flesh, with larger stone cells concentrated around the core and smaller ones in the pericarp. There were no obvious differences in stone cell structure between the two varieties, but stone cell size and content was much greater in’Dangshansuli’than in’Kousui.’Further, lignin accounted for 29.8% of stone cell composition in’Dangshansuli’, a significantly higher proportion than that in ’Kousui’(24.6%), basing on acetyl bromide method; this result was confirmed by autofluorescence analysis. More detailed information on lignin distribution across the cell wall was obtained by TEM combined with the KMnO4 staining technique. In TEM images, cell walls of both pear varieties were typically divided into four layers:compound middle lamella (CML), secondary wall 1 (S1), outer secondary wall 2 (S2L) and secondary wall 2 (S2), with different staining intensities for different lignin concentrations in those regions.2. Lignin isolated from sclereids of’Dangshansuli’and’Kousui’pear were studied with several spectroscopies, such as ultra violet spectroscopy (UV), fourier transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC),1H nuclear magnetic resonance (1H-NMR) and 13C nuclear magnetic resonance (13C-NMR). The lignin were the same in structural information, however the weight-average molecular weight (WMW) and molecular weight distribution (MWD) of’Kousui’lignin both were higher than ’Dangshansuli’lignin, consequently, it had higher glass transition temperature (GTT). Typical benzene structure showed in UV spectra was also confirmed by IR spectra. Great vanillin and some syringaldehyde found in lignin nitrobenzene oxidation products and Maule reagent staining result indicated the presence of guaiacyl unit in the lignin. Further, with the aids of 1H and 13C-NMR spectra, lignin in pear sclereids was characterized as typical guaiacyl-syringyl lignin (G-S lingnin), however it has higher content guaiacyl unit than other angiosperm, the ratio for guaiacyl and syringyl unit was as high as 4, more closely to gymnosperm.3. Distribution density of sclereids was highest in pear fruitlet and then descended with fruit development; however the size of sclereid cluster increased gradually till fruit mature. With regards to phenylalanine ammonia lyase (PAL), peroxidase (POD) and polyphenol oxidase (PPO), their activity peaks always appeard in fruitlets, and then declined gradually in the course of fruit development. Fruit bagging had good effects on’Dangshansuli’fruit quality; it can reduce the content and size of sclereid in mature fruit; furthermore, bagging stage had influence on the effects, the earlier the better. Also, CaCl2 showed the same effects as fruit bagging on both’Dangshansuli’and’Kousui’, while PP333 had the opposite result. All the data from this section showed a positive relationship between the enzymes’ activities and lignin, sclereid content and size. Especially the cell wall bounded peroxidase (PODⅡ) played more important role in cell lignification.4. The relationship among peroxidase, hydrogen peroxide, lignin and sclereid was studied by using’Dangshansuli’as material. Sclereids accounted for a big amount in dry fruit weight as 36.03%, and they contained 29.83%lignin which accounted for 70.6%of the total in fruit. Peroxidase activity was found almost around sclereids in free hand sections, and in the cell wall at cellular level. Besides, hydrogen peroxide was also localized at the similar regions, such as cell wall, cell corner and intercellular layer. Lignin was synthesized in big scale along with a burst of hydrogen peroxide in young fruit; also the soluble peroxidase (PODⅠ) and cell wall bounded peroxidase (PODⅡ) showed the same changing tendency, however the PODⅡalways possessed higher affinity to syringaldazine-an analog of lignin monomer. Further study basing on substrate affinity analysis demonstrated that it was the basic PODⅡshowing high catalytic efficiency. Lignin biosynthesis and sclereid development were related to basic PODⅡ.5. Two PAL genes, two POD genes, and two CAD genes were cloned and sequenced using RT-PCR from’Dangshansuli’and’Kousui’pear respectively. In nucleotide sequence, the two PAL genes had 3 bp differences, and coded an identical amino acid sequence which comprised of 217 residues. Meanwhile, the amino acid sequence of the two PAL was 98.6%identical to PAL from Pyrus communis (ABB70117),98.6% identical to PAL from Prunus avium (AAC78457),97.7%identical to PAL from Rubus idaeus (AAF40224), and 97.2%indentical to PAL from Citrus clementina×Citrus reticulata (CAB42793) respectivity. Further, they were found to have 4 similar active sites with PAL from other plant species.The two CAD genes had 4 bp differences resuting in one residue differences. The deduced amino acid sequence of the two CAD was 97.8%identical to CAD from Malus domestica (AAC06319),95%identical to CAD from Prunus mume (BAE48658), 92.6%identical to CAD from Eriobotrya japonica (ABV44810) and 83.4%identical to CAD from Vitis vinifera (CAO21890) respectively.The two POD genes were identical in both nucleotide sequences and deduced amino acid sequences. Deduced amino acid sequence of the POD from pear was 84.3% identical to POD from Nicotiana tabacum (AAD33072),86.4%identical to POD from Oncidium Gower Ramsey (ABC02343),86.9%identical to POD from Thellungiella halophila (ABU54828) and 86.4%identical to POD from Arabidopsis thaliana (CAA6686) respectively. Moreover, POD from these two pears was found to have 3 similar active sites and 2 substrate bounding sites with POD from other plant species.All the results from above showed PB-PAL (Accession No. FJ478149), PP-PAL (Accession No. FJ478150), PB-CAD (Accession No. FJ478151), PP-CAD (Accession No. FJ478152), PB-POD (Accession No. FJ478153) and PP-POD (Accession No. FJ478154) were the PAL, CAD and POD genes in pear fruit.