- Genome-wide identification and expression analyses of the pectate lyase ( PEL ) gene. - Background: Pectin is a major component and structural polysaccharide of the primary cell walls and middle lamella of higher plants. - Nevertheless, there have been few studies on PEL genes and no comprehensive analysis of the PEL gene family in cotton.. - expansion of the GhPELs . - 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - In pollen de- velopment, PELs may regulate the loosening and deg- radation of the pollen cell wall [11]. - In the process of fruit ripening, PELs modify the pectin structure in the cell wall [13]. - In Populus , most of the 30 PtPLs are highly expressed in xylem, performing important functions during the development of wood [17]. - Auxin, an important plant hormone, regulates plant growth and development by advancing acid-mediated changes in the cell wall [21, 22].. - The acidification of the cell wall activates the expansins and PMEs, which causes loosening of the cell wall [2].. - Pec- tins are responsible for 25% of the cell wall components of rapidly elongating cotton fibers (8 days postanthesis (DPA. - A study examining GhPEL indicated that this gene is crucial for the normal elongation of cotton fibers through degrad- ation of de-esterified pectin, facilitating the loosening of the cell wall [16].. - In cotton, most of the PEL genes are unknown. - At present, there are no available genome-wide analyses of the cotton PEL gene family.. - With the completion of the genome sequencing of G. - According to their locations on the chromosomes, the family members of the three species were designated GrPEL1 to GrPEL53 . - The lengths of the putative GhPEL proteins varied from 171 (GhPEL20_At) to 680 (GhPEL52_Dt) amino acids (aa), while those of GaPELs ranged from 136 aa (GaPEL36) to 680 aa (GaPEL14), and those GrPELs var- ied from 222 aa (GrPEL36) to 511 aa (GrPEL27). - The predicted Mw, pI, GRAVY and subcellular localization of the protein sequences are shown in Additional file 1:. - Phylogenetic analysis of the PEL gene family. - To examine the evolutionary relationships of the PEL proteins and classify them into subfamilies according to the established subfamilies in Arabidopsis, we performed a phylogenetic analysis of 285 PEL protein sequences from G. - The PEL proteins of Oryza sativa , a monocot, were distant from the PEL proteins of the other dicot plants. - In the present study, a gene duplication analysis was per- formed to investigate the expansion mechanism of the PEL gene family in the three Gossypium species. - hirsutum , respectively, accounting for and 86.7% of the PEL gene family (Additional file 4: Table S4). - These re- sults indicated that gene duplication, especially segmental duplication, played an irreplaceable role in the expansion of the PEL gene family in the three Gossypium species.. - The results showed that the Ka/Ks ratios for most of the segmental duplications of PEL gene pairs were less than 1.0, indicating that they had experienced puri- fying selection pressure after gene duplication events (Additional file 5: Table S5). - Because of the constraints. - of purifying selection on divergence, most of the seg- mental duplications of the PEL pairs might exhibit simi- lar functions. - arboreum , the timing of the occurrence of the segmental duplication of PEL pairs was inferred to be million years ago (MYA), with an average of 91.73 MYA, and MYA, with an average of 85.73 MYA, respectively. - hirsutum , the timing of the occur- rence of the segmental duplication PEL pairs was pre- sumed to be MYA, with an average of 10.24 MYA.. - Conserved domains and amino acid sites of GhPELs The Pec_lyase_C domains and signal peptides of the PEL sequences were investigated and shown according to the phylogenetic tree of the GhPELs (Additional file 6:. - All of the GhPELs contained a Pec_lyase_C domain, indicating that this domain was conserved. - Most members of the GhPELs (77.1%) exhibited the predicted signal peptide. - Gene structure and conserved protein motifs of GhPELs To further understand the conservation and diversifica- tion of the GhPELs , their exon-intron structures and conserved motifs were investigated and were shown in Fig. - We identified 6 conserved motifs of the GhPEL pro- teins using MEME (Fig. - Within a given subfamily, most of the members exhibited similar motif construc- tion. - Motifs 1, 5, 3 and 6 within the conserved Pec_lya- se_C domain were identified in most of the GhPEL proteins. - A total of 90.4% of the GhPEL proteins (except for the members of subfamily I and GhPEL20_At, GhPEL40_At, GhPEL19_At, and GhPEL43_Dt) con- tained motif 4. - Motif 2 existed in 96.4% of the GhPEL proteins (except for GhPEL20_At, GhPE- L40_At, and GhPEL43_Dt). - and their arrangement showed a high conservation in the GhPEL family.. - The results showed that all of the putative GhPEL promoter regions contained at least one of the six major auxin-responsive cis-elements:. - A Ca 2+ -responsive cis-element (S000501) and calmodulin-binding/CGCG box (S000507) were identified in of the. - This result indicated that some GhPELs might alter the configuration of the cell wall with Ca 2+. - hirsutum (TM-1) [30], the expression pro- files of 62 GhPELs with FPKM ≥1 in at least one of the 8 investigated tissues were shown in Fig. - The other 21 GhPELs , including all of the members (12) of the subfamily II, were very low or not expressed in all of the investigated tissues and developmental stages and 15 genes from gene duplication events, in- dicating that functional redundancy or pseudogenes existed in the GhPEL family.. - general expression in all of the tissues, with some genes primarily expressed in fiber, stamen and other tissues.. - The dominant expression of GhPELs in cluster A indicated that these genes performed crucial and conserved func- tions in the development of the flower, especially the stamen, which was concordant with PLLs generally expressed in flower and several PLLs highly expressed in pollen in Arabidopsis [19]. - b Exon-intron organization of the GhPEL family. - In the present study, a. - a-c: High expression in the meiosis stage, binucleate stage and mature stage of anthers. - In our study, analysis of the timing of the occurrence of seg- mental duplications showed that most of the events ob- served in G. - 8 Expression analysis of GhPELs in the leaves under IAA treatment via qRT-PCR. - hirsutum , the segmental duplication events most likely occurred before the hybridization of the two extant ancestors, ac- cording to the range of divergence times MYA) and average time (10.24 MYA). - The uneven chromosomal distribution of the PEL genes in the three cotton species might be due to the occurrence of these gene duplication events in the evolution of cotton.. - The PELs of Oryza sativa were located a long distance from those of the other plants, which might be related to different functions of these proteins between monocots and dicots [39]. - All of the GhPELs exhibited the conserved Pec_lyase_C domain, with highly conserve Ca 2. - The conserved motif analysis showed that the six motifs existed in most of the GhPELs (Fig. - The similar- ities and differences of the gene structures, domains and motifs of GhPELs might be related to conservation and subfunctionalization, resulting from their long evolution- ary history and gene duplication in cotton [37, 41].. - PELs are cell wall-modifying enzymes that can cleave α-1,4-glycosidic linkages of demethylated HG, the struc- tural polysaccharide of the primary cell walls and middle lamella in higher plants, via a β-elimination mechanism [5]. - Demethylated HG is degraded by PELs and PGs more easily, promoting the loosening of the cell wall and regulating cell growth, cell division and organ morphogenesis in plant growth and development . - In the present study, a transcriptomic analysis of GhPELs revealed that 90% of the members in subfamily IV were preferentially expressed in the sta- men, in agreement with the above reports (Fig. - To further explore the differences in the expression of. - The PELs expressed during anther development might function to facilitate the degradation of the primary cell wall in pollen mother cells (PMC) during the meiosis stage and take part in anther dehiscence, cell wall loos- ening in pollen, pollen tube elongation and the promo- tion of pollen penetration through style tissue degradation in mature anthers . - Cotton fiber, which is a widely used natural fiber in the textile industry, is produced from a single differ- entiated epidermal cell of the ovule [47, 48]. - According to our analysis of cis-elements, all of the GhPELs contained at least one of the six auxin-responsive cis-elements (Additional file 8: Table S6). - However, the regulatory networks and functions of the GhPELs require further studies.. - We performed a genome-wide analysis of the PEL gene family in G. - All of the GhPELs exhibited the conserved Pec_lyase_C domain, and diverse gene structures were observed among them. - Then, the normal mode of the SMART database (http://smart.embl-heidelberg.de/) was used to confirm every putative GhPEL protein with a Pec_lya- se_C domain [57]. - The theoretical molecular weight (Mw), isoelectric point (pI), grand average of hydropathicity (GRAVY) and sub- cellular localization of the predicted GhPELs were pre- dicted using ExPASy (http://cn.expasy.org/tools) and the CELLO v2.5 server (http://cello.life. - The physical chromosome locations of all PEL genes were obtained from the genome sequence databases of the three Gossypium species and visualized with MapIn- spect (http://www.plantbreeding.wur.nl/uk/ software- mapinspect.html). - The predicted PEL proteins of the three cotton species were aligned with ClustalW2 at EMBL-EBI (http://www.ebi.ac.uk/Tools/msa/clustalw2/).. - Gene duplication was confirmed if the following condi- tions were satisfied: (1) the coverage of the alignment was >. - 80% of the longer gene. - (2) the identity of the. - Nonsynonymous (Ka) and synonymous substitution (Ks) rates were calculated using DnaSp V5.0 software, employing the full-length gene sequences of the segmen- tal duplicated PEL gene pairs from the three cotton spe- cies aligned by ClustalX 2.0 [66]. - The genomic sequences and positions of the exons and introns of GhPELs were employed to visualize PEL exon-intron structures on the Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn/) [69]. - The MEME program was used to analyze the conserved motifs of the GhPEL protein sequences with the following parameters: site distribution, zero or one occurrence per sequence. - To determine the cis-elements of the predicted pro- moters, the 2000 bp genomic DNA sequences upstream of the initiation codon (ATG) of all GhPELs were employed to search the PLACE database (http://. - The details of the protocol were as follows: (Step 1) initial denaturation step of 30 s at 95 °C, (Step 2) 40 cycles of 5 s at 95 °C, 34 s at 60 °C and (Step 3) melting curve analysis. - Detailed parameters of the PEL proteins from three cotton species. - Numbers of PELs in the five subfamilies in different species. - Conserved amino acid sites of the GhPEL proteins. - Cis-elements related to auxin in the promoters of GhPEL genes. - This study was performed at the State Key Laboratory of Cotton Biology at the Institute of Cotton Research of the Chinese Academy of Agricultural Sciences.. - The funders had no role in the design of the study, the collection, analysis, and interpretation of data, and in writing the manuscript.. - Molecular and genetic characterization of two pollen-expressed genes that have sequence similarity to pectate lyases of the plant pathogen Erwinia . - Identification of the tobacco and Arabidopsis homologues of the pollen-expressed LAT59 gene of tomato. - The essential role of GhPEL gene, encoding a pectate lyase, in cell wall loosening by depolymerization of the de-esterified pectin during fiber elongation in cotton. - Characterization and functional analysis of the poplar Pectate Lyase-like gene PtPL1-18 reveal its role in the development of vascular tissues. - Analysis of promoter activity of members of the PECTATE LYASE-LIKE (PLL) gene family in cell separation in Arabidopsis.. - Growth of the plant cell wall. - Patterns of auxin transport and gene expression during primordium development revealed by live imaging of the Arabidopsis inflorescence meristem. - Changes in biochemical composition of the cell wall of the cotton fiber during development. - Genome sequence of the cultivated cotton Gossypium arboreum. - Genome-wide characterization of the Pectate Lyase-like (PLL) genes in Brassica rapa. - Genome-wide characterization and comparative analysis of the MLO gene family in cotton. - Tetrad pollen formation in quartet mutants of Arabidopsis thaliana is associated with persistence of pectic polysaccharides of the pollen mother cell wall. - A genome-wide analysis of the small auxin-up RNA (SAUR) gene family in cotton. - Wei F, Coe E, Nelson W, Bharti AK, Engler F, Butler E, Kim H, Goicoechea JL, Chen M, Lee S et al: Physical and Genetic Structure of the Maize Genome Reflects its Complex Evolutionary History. - Divergence of the Dof gene families in poplar, Arabidopsis, and rice suggests multiple modes of gene evolution after duplication
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