Genome-wide identification and functional analysis of U-box E3 ubiquitin ligases gene family related to drought stress response in Chinese white pear (Pyrus bretschneideri).

BACKGROUND: The plant U-box (PUB) proteins are a family of ubiquitin ligases (E3) enzymes that involved in diverse biological processes, as well as in responses to plant stress response. However, the characteristics and functional divergence of the PUB gene family have not yet been previously studied in the Chinese white pear (Pyrus bretschneideri). RESULTS: In the present study, we identified 62 PbrPUBs in Chinese white pear genome. Based on the phylogenetic relationship, 62 PUB genes were clustered into five groups. The results of conserved motif and gene structure analysis supported the classification phylogenetic tree. The PbrPUB genes were unevenly distribution on 17 pear chromosomes, chromosome 15 housed most member of PUB family, with eight PUB genes. Cis-acting element analysis indicated that PUB genes might participate in diverse biological processes, especially in the response to abiotic stresses. Based on RNA-data from 'Dangshansuli' at seven tissues, we found that PUB genes exhibited diverse of expression level in seven tissues, and qRT-PCR experiment further supported the reliable of RNA-Seq data. To identify candidate genes associated with resistance, we conducted qRT-PCR experiment the expression level of pear seed plant under four abiotic stresses, including: ABA, dehydration, salt and cold treatment. One candidate PUB gene associated with dehydration stress was selected to conduct further functional experiment. Subcellular localization revealed PbrPUB18 protein was located on cell nucleus. Furthermore, heterologous over-expression of PbrPUB18 in Arabidopsis indicated that the over-expression of PbrPUB18 could enhance resistance in drought treatment. In conclusions, we systematically identified the PUB genes in pear, and provided useful knowledge for functional identification of PUB genes in pear.

Efficacy of postharvest sodium nitroprusside application to extend storability by regulating physico-chemical quality of pear fruit.

Quality loss in pear fruit during storage reduces its marketability for long run. To increase its storability, the efficacy of postharvest dip treatment donor sodium nitroprusside (SNP) 0.000, 0.001, 0.002 and 0.003 mol L-1 were investigated on pear fruit cv. Patharnakh under storage conditions (low temperature 0-1 °C and relative humidity (90-95%). SNP effectively lowered fruit mass loss, retained colour and higher firmness, suppressed browning and respiration rate and sustained soluble solids content, titratable acidity, total phenol content and ascorbic acid thus conserved the fruit quality for longer period. SNP treatments suppressed the activity of polyphenol oxidase and increased activity of superoxide dismutase enzyme. Additionally, the SNP treated fruit exhibited lesser activities of fruit softening enzymes like pectin methylesterase, polygalacturonase and cellulase. Among all, 0.002 mol L-1 SNP concentration was superior to lengthen storability and sensory quality of pear up to 60 d under cold storage.

Cloning and functional characterization of two cinnamate 4-hydroxylase genes from Pyrus bretschneideri.

Cinnamate 4-hydroxylase (C4H) is a key enzyme in the phenylpropanoid pathway in plants and is involved in the biosynthesis of secondary metabolites such as lignin and flavonoids. However, the function of C4H in pear plants (Pyrus bretschneideri) has not yet been fully elucidated. By searching pear genome databases, we identified three C4H genes (PbC4H1, PbC4H2 and PbC4H3) encoding proteins that share higher identity with bonafide C4Hs from several species with typical cytochrome P450 domains, suggesting that all three PbC4Hs are also bonafide C4Hs that have close evolutionary relationships with C4Hs from other land plants. Quantitative real-time PCR (qRT-PCR) results indicated that the three PbC4Hs were specifically expressed in one or more tissues. The expression levels of PbC4H1 and PbC4H3 first increased and then decreased during pear fruit development. Treatment with exogenous hormones (ABA, MeJA, and SA) altered the expression of the three PbC4Hs to varying degrees. The expression levels of the PbC4Hs were first induced and then decreased under ABA treatment, while MeJA treatment significantly increased the expression levels of the PbC4Hs. Following treatment with SA, expression levels of PbC4H1 and PbC4H2 increased, while expression levels of PbC4H3 decreased. Enzymatic analysis of the recombinant proteins expressed in yeast indicated that PbC4H1 and PbC4H3 catalysed the conversion of trans-cinnamic acid to p-coumaric acid. Moreover, the expression of PbC4H1 and PbC4H3 in Arabidopsis resulted in an increase in both the lignin content and the thickness of cell walls for intervascular fibres and xylem cells. Taken together, the results of our study not only revealed the potential role of PbC4H1 and PbC4H3 in lignin biosynthesis but also established a foundation for future investigations of the regulation of lignin synthesis and stone cell development in pear fruit by molecular biological techniques.

Genome-wide characterization of the cellulose synthase gene superfamily in Pyrus bretschneideri and reveal its potential role in stone cell formation.

Members of the cellulose synthase (CesA) and cellulose synthase-like (Csl) families from the cellulose synthase gene superfamily participate in cellulose and hemicellulose synthesis in the plasma membrane. The members of this superfamily are vital for cell wall construction during plant growth and development. However, little is known about their function in pear fruit, a model for Rosaceae species and for fleshy fruit development. In our research, a total of 36 CesA/Csl family members were identified from the pear and were grouped into six subfamilies (CesA, CslB, CslC, CslD, CslE, and CslG) according to phylogenetic relationships. We performed a protein sequence physicochemical analysis, phylogenetic tree construction, a gene structure, a conserved domain, and chromosomal localization analysis. The results indicated that most of the CesA/Csl genes from pear are closely related to genes in Arabidopsis, but these families have unique characteristics in terms of their gene structure, chromosomal localization, phylogeny, and deduced protein sequences, suggesting that they have evolved through different processes. Tissue expression analysis results showed that most of the CesA/Csl genes were constitutively expressed at different levels in different organs. Furthermore, the expression levels of four genes (Pbr032894.2, Pbr016107.1, Pbr00518.1, and Pbr034218.1) tended to first increase and then decrease during fruit development, implying that these four genes may be involved in the development of stone cells of pear fruit. Our results may help elucidate the evolutionary history and functional differences of the CesA/Csl genes in pear and lay a foundation for further investigation of the CesA/Csl genes in pear and other Rosaceae species.

Gene structure, evolution and expression analysis of the P-ATPase gene family in Chinese pear (Pyrus bretschneideri).

P-ATPase are a large protein family of integral membrane, playing an important role in plant growth, development and stress. P-ATPase genes family have been identified and characterized in several model plants such as cotton, grapes, tobacco, rice, rubber plant and Arabidopsis. However, still lack of comprehensive study of P-ATPase genes in Chinese pear (Pyrus bretschneideri). A systematic analysis was performed and identified 30 P-ATPase genes from the pear genome to evaluate the qualities and diversity of P-ATPase proteins. Phylogenetic analysis was performed using A. thaliana P-ATPase genes as a model, allowing us to categorize into 4 subfamilies (PbHMA, PbECA, PbACA, and PbAHA) and two subfamilies (ALA and P5) is absent in pear. Even Within the same subclade, P-ATPase genes also shows the similar exon-intron structure and conserved motif structure. Continuing chromosomal localization analysis showed that 23 P-ATPase genes were distributed among 13 chromosome and 7 gene on the scaffold of pear. Promoter regions of P-ATPase genes revealed that several cis-acting elements were involved in plant growth/development, stress responses as well as hormone responses. Additionally, P-ATPase genes were also differentially expressed under hormones treatments of ABA (abscisic acid) and SA (salicylic acid) treatments. Remarkably, the transcriptome data exposed that P-ATPase gene might play an important role in lignin biosynthesis during fruit development. The real time qRT-PCR was performed, and the expression analysis indicated that various P-ATPase genes extremely expressed during different developmental stages of fruit. Our study provides valuable information about the P-ATPase gene family in pear fruit development and lignin polymerization.

Genome-wide identification and comparative analysis of the ADH gene family in Chinese white pear (Pyrus bretschneideri) and other Rosaceae species.

Alcohol dehydrogenase (ADH) is essential to the formation of aromatic compounds in fruits. However, the evolutionary history and characteristics of ADH gene expression remain largely unclear in Rosaceae fruit species. In this study, 464 ADH genes were identified in eight Rosaceae fruit species, 68 of the genes were from pear and which were classified into four subgroups. Frequent single gene duplication events were found to have contributed to the formation of ADH gene clusters and the expansion of the ADH gene family in these eight Rosaceae species. Purifying selection was the major force in ADH gene evolution. The younger genes derived from tandem and proximal duplications had evolved faster than those derived from other types of duplication. RNA-Seq and qRT-PCR analysis revealed that the expression levels of three ADH genes were closely correlated with the content of aromatic compounds detected during fruit development.

Acid vacuolar invertase 1 (PbrAc-Inv1) and invertase inhibitor 5 (PbrII5) were involved in sucrose hydrolysis during postharvest pear storage.

In pear, sucrose was mainly distributed in vacuole; and the alternation of sucrose abundance was associated the change of vacuolar invertase (VI) activity during fruit storage. However, the molecular mechanism beneath such phenomenon has not been clarified until recently. For this, a combination of metabolite, enzyme activity, transcriptome, quantitative real-time PCR (qRT-PCR), bioinformation, subcellular localization, and transient overexpression assay was conducted in this study to identify the acid invertase 1 (PbrAc-Inv1) and invertase inhibitor 5 (PbrII5) involved in sucrose degradation during 'Housui' pear storage. Both PbrAc-Inv1 and PbrII5 were located in vacuolar membrane. PbrAc-Inv1 could accelerate sucrose degradation; on the other hand, PbrII5 could bind with PbrAc-Inv1 to form a inactive complex, downregulate the VI activity, and suppressed sucrose decomposition. Based on Bio-layer interferometry (BLI) result after domain substitution, the domain on the left of catalytic 'WEC-P/V-D' box in PbrAc-Inv1 might played a key role in its interaction with PbrII5.

Characterization of the pectin methyl-esterase gene family and its function in controlling pollen tube growth in pear (Pyrus bretschneideri).

Pectin methyl-esterases (PMEs) play crucial roles in plant growth. In this study, we identified 81 PbrPMEs in pear. Whole-genome duplication and purifying selection drove the evolution of PbrPME gene family. The expression of 47 PbrPMEs was detected in pear pollen tube, which were assigned to 13 clusters by an expression tendency analysis. One of the 13 clusters presented opposite expression trends towards the changes of methyl-esterified pectins at the apical cell wall. PbrPMEs were localized in the cytoplasm and plasma membrane. Repression of PbrPME11, PbrPME44, and PbrPME59 resulted in the inhibition of pear pollen tube growth and abnormal deposition of methyl-esterified pectins at pollen tube tip. Pharmacological analysis confirmed that reduced PbrPME activities repressed the pollen tube growth. Overall, we have explored the evolutionary characteristics of PbrPME gene family and found the key PbrPME genes that control the growth of pollen tube, which deepened the understanding of pear fertility regulation.

Genome-wide analysis of polygalacturonase gene family from pear genome and identification of the member involved in pear softening.

BACKGROUND: Polygalacturonase (PG), as an important hydrolase participating in the degradation of pectin, plays an important role in softening process of fruit. However, information on PG gene family in pear genome and the specific member involved in fruit softening is still rudimentary. RESULTS: In this study, a total of 61 PG genes, which could be divided into six subclasses, were identified from the pear genome with diverse chromosome locations, gene structures, motifs and cis-acting elements. Most PbrPGs were derived from WGD/segmental duplication blocks, and purifying selection was the main driving force for their expansion. The expression profiles of PbrPGs in pear were tissue/development-stage/cultivar-dependent. During 'Housui' pear storage, associated with the reduction of firmness was the accumulation of PG activity. Totally, 28 PbrPGs were expressed during fruit storage, which could be classified into five categories based on different expression patterns; most demonstrated an increased trend. Of these, PbrPG6 were proposed to account for pear softening in combination of the phylogenetic and correlation analysis among firmness, PG activity and PbrPGs. By constructing the silencing vector, a higher firmness was observed in PbrPG6-silenced fruit when compared with that of the control (empty vector). In a further study, we found that the expression of PbrPG6 was regulated by postharvest 1-MCP/ethrel treatment, and several PbrERFs might function in this process. CONCLUSIONS: We identified 61 PbrPG genes from pear genome; of these, PbrPG6 was involved in fruit softening process; furthermore, the expression of PbrPG6 might be under the control of PbrERF. This study provides a foundation for future work aimed at elucidating the molecular mechanism underlying pear softening.

The ß-amylase PbrBAM3 from pear (Pyrus betulaefolia) regulates soluble sugar accumulation and ROS homeostasis in response to cold stress.

ß-Amylase (BAM) is involved in sugar metabolism, but the role of BAM genes in cold tolerance remains poorly understood. Here, we report the identification and functional characterization of the chloroplast-localized BAM-encoding gene PbrBAM3 isolated from Pyrus betulaefolia. The transcript levels of PbrBAM3 were up-regulated under cold, dehydration and ABA, but repressed by maltose. Overexpression of PbrBAM3 in tobacco (Nicotiana tabacum) and pear (P. ussuriensis) conferred increased BAM activity, promoted starch degradation after chilling treatments and enhanced tolerance to cold. Under the chilling stress, the transgenic tobacco and P. ussuriensis exhibited lessened reactive oxygen species (ROS) generation, higher levels of antioxidant enzymes activity, and greater accumulation of soluble sugars (specially maltose) than the corresponding wild type plants. Taken together, these results demonstrate that PbrBAM3 plays an important role in cold tolerance, at least in part, by raising the levels of soluble sugars capable of acting as osmolytes or antioxidants.

Identification of hexokinase family members in pear (Pyrus × bretschneideri) and functional exploration of PbHXK1 in modulating sugar content and plant growth.

Hexokinase (HXK) is a multifunctional protein that serves as a sugar sensor for glucose signaling and a catalyst for glycolysis. It has been well studied in many species, however, there is far less information about this family in pear. To investigate the roles of HXK in the growth and development of pear fruit, we performed a genome-wide analysis and identified the HXK gene family members in pear. In addition, we functionally characterized a glucose sensor gene, PbHXK1, in P. bretschneideri. In total, 10 HXK genes were identified in pear, and a multiple sequence alignment and phylogenetic analysis showed that PbHXK1 is a Type B HXK that contains four conserved domains, phosphate 1 and 2, sugar binding and adenosine, which are specific to plant HXKs and essential for enzymatic functions. A qRT-PCR analysis revealed that the relative expression levels of PbHXK1 were negatively correlated with sugar content but significantly positively correlated with HXK activity during pear fruit development. Furthermore, the overexpression of PbHXK1 in tomatoes significantly enhanced the HXK activity and decreased the sugar content. In addition, the growth of transgenic tomato plants overexpressing PbHXK1 was inhibited, leading to shortened internodes and smaller leaves. Thus, in pear, PbHXK1 encodes HXK, which regulated the sugar content in fruit and affected the growth and development of plants.

In Silico Genome-Wide Analysis of Respiratory Burst Oxidase Homolog (RBOH) Family Genes in Five Fruit-Producing Trees, and Potential Functional Analysis on Lignification of Stone Cells in Chinese White Pear.

: The accumulation of lignin in fruit has a significant negative impact on the quality of fruit-producing trees, and in particular the lignin formation stimulates the development of stone cells in pear fruit. Reactive oxygen species (ROS) are essential for lignin polymerization. However, knowledge of the RBOH family, a key enzyme in ROS metabolism, remains unknown in most fruit trees. In this study, a total of 40 RBOHs were identified from five fruit-producing trees (Pyrusbretschneideri, Prunuspersica, Citrussinensis, Vitisvinifera, and Prunusmume), and 10 of these sequences came from Pyrusbretschneideri. Multiple sequence alignments revealed that all 10 PbRBOHs contained the NADPH_Ox domain and the six alpha-helical transmembrane domains (TM-I to TM-VI). Chromosome localization and interspecies phylogenetic tree analysis showed that 10 PbRBOHs irregularly distributed on 8 chromosomes and 3 PbRBOHs (PbRBOHA, PbRBOHB, and PbRBOHD) are closely related to known lignification-related RBOHs. Furthermore, hormone response pattern analysis showed that the transcription of PbRBOHs is regulated by SA, ABA and MeJA. Reverse transcription-quantitative real-time polymerase chain reaction (qRT-PCR) and transcriptome sequencing analysis showed that PbRBOHA, PbRBOHB, and PbRBOHD accumulated high transcript abundance in pear fruit, and the transcriptional trends of PbRBOHA and PbRBOHD was consistent with the change of stone cell content during fruit development. In addition, subcellular localization revealed that PbRBOHA and PbRBOHD are distributed on the plasma membrane. Combining the changes of apoplastic superoxide (O2.-) content and spatio-temporal expression analysis, these results indicate that PbRBOHA and PbRBOHD, which are candidate genes, may play an important role in ROS metabolism during the lignification of pear stone cells. This study not only provided insight into the molecular characteristics of the RBOH family in fruit-producing trees, but also lays the foundation for studying the role of ROS in plant lignification.

PbrSLAH3 is a nitrate-selective anion channel which is modulated by calcium-dependent protein kinase 32 in pear.

BACKGROUND: The functional characteristics of SLAC/SLAH family members isolated from Arabidopsis thaliana, poplar, barley and rice have been comprehensively investigated. However, there are no reports regarding SLAC/SLAH family genes from Rosaceae plants. RESULTS: In this study, the function of PbrSLAH3, which is predominately expressed in pear (Pyrus bretschneideri) root, was investigated. PbrSLAH3 can rescue the ammonium toxicity phenomenon of slah3 mutant plants under high-ammonium/low-nitrate conditions. In addition, yeast two-hybrid and bimolecular fluorescence complementation assays confirmed that PbrSLAH3 interacts with PbrCPK32. Moreover, when PbrSLAH3 was co-expressed with either the Arabidopsis calcium-dependent protein kinase (CPK) 21 or PbrCPK32 in Xenopus oocytes, yellow fluorescence was emitted from the oocytes and typical anion currents were recorded in the presence of extracellular NO3-. However, when PbrSLAH3 alone was injected, no yellow fluorescence or anion currents were recorded, suggesting that anion channel PbrSLAH3 activity was controlled through phosphorylation. Finally, electrophysiological and transgene results showed that PbrSLAH3 was more permeable to NO3- than Cl-. CONCLUSION: We suggest that PbrSLAH3 crossing-talk with PbrCPK32 probably participate in transporting of nitrate nutrition in pear root.

Genome-wide identification, expression and functional analysis of the phosphofructokinase gene family in Chinese white pear (Pyrus bretschneideri).

Phosphofructokinase plays an essential role in sugar metabolism in plants. Plants possess two types of phosphofructokinase proteins for phosphorylation of fructose-6-phosphate, the pyrophosphate-dependent fructose-6-phosphate phosphotransferase (PFP), and the ATP-dependent phosphofructokinase (PFK). Until now, the gene evolution, expression patterns, and functions of phosphofructokinase proteins were unknown in pear. In this report, 14 phosphofructokinase genes were identified in pear. The phylogenetic tree indicated that the phosphofructokinase gene family could be grouped into two subfamilies, with 10 genes belonging to the PbPFK subfamily, and 4 genes belonging to the PbPFP subfamily. Conserved motifs and exon numbers of the phosphofructokinase were found in pear and other six species. The evolution analysis indicated that WGD/Segmental and dispersed duplications were the main duplication models for the phosphofructokinase genes expansion in pear and other six species. Analysis of cis-regulatory element sequences of all phosphofructokinase genes identified light regulation and the MYB binding site in the promoter of all pear phosphofructokinase genes, suggesting that phosphofructokinase might could be regulated by light and MYB transcription factors (TFs). Gene expression patterns revealed that PbPFP1 showed similar pattern with sorbitol contents, suggesting important contributions to sugar accumulation during fruit development. Further functional analysis indicated that the phosphofructokinase gene PbPFP1 was localized on plasma membrane compartment, indicating that PbPFP1 had function in plasma membrane. Transient transformation of PbPFP1 in pear fruits led to significant increases of fructose and sorbitol compared to controls. Overall, our study provides important insights into the gene expression patterns and important potential functions of phosphofructokinase for sugar accumulation in pear fruits, which will help to enrich understanding of sugar-related bio-pathways and lay the molecular basis for fruit quality improvement.

Comprehensive genome-wide analysis of the pear (Pyrus bretschneideri) laccase gene (PbLAC) family and functional identification of PbLAC1 involved in lignin biosynthesis.

The content and size of stone cell clusters affects the quality of pear fruit, and monolignol polymerization and deposition in the cell walls constitute a required step for stone cell formation. Laccase (LAC) is the key enzyme responsible for the polymerization of monolignols. However, there are no reports on the LAC family in pear (Pyrus bretschneideri), and the identity of the members responsible for lignin synthesis has not been clarified. Here, 41 LACs were identified in the whole genome of pear. All Pyrus bretschneideri LACs (PbLACs) were distributed on 13 chromosomes and divided into four phylogenetic groups (I-IV). In addition, 16 segmental duplication events were found, implying that segmental duplication was a primary reason for the expansion of the PbLAC family. LACs from the genomes of three Rosaceae species (Prunus mummer, Prunus persica, and Fragaria vesca) were also identified, and an interspecies collinearity analysis was performed. The phylogenetic analysis, sequence alignments and spatiotemporal expression pattern analysis suggested that PbLAC1, 5, 6, 29, 36 and 38 were likely associated with lignin synthesis and stone cell formation in fruit. The two target genes of Pyr-miR1890 (a microRNA identified from pear fruit that is associated with lignin and stone cell accumulation), PbLAC1 and PbLAC14, were selected for genetic transformation. Interfamily transfer of PbLAC1 into Arabidopsis resulted in a significant increase (approximately 17%) in the lignin content and thicker cell walls in interfascicular fibre and xylem cells, which demonstrated that PbLAC1 is involved in lignin biosynthesis and cell wall development. However, the lignin content and cell wall thickness were not changed significantly in the PbLAC14-overexpressing transgenic Arabidopsis plants. This study revealed the function of PbLAC1 in lignin synthesis and provides important insights into the characteristics and evolution of the PbLAC family.

Influence of Nucleophilic Amino Acids on Enzymatic Browning Systems.

In the present study the enzymatic oxidation of gallic acid and catechin catalyzed by nashi pear polyphenol oxidase (PPO) in the presence of the amino acids lysine, arginine, or cysteine was investigated for polyphenol-amino acid adducts. HPLC analyses revealed the formation of two novel dihydrobenzothiazine carboxylic acid derivatives (8-(3',4'-dihydro-2 H-chromene-3',5',7'-triol)-3,4-dihydro-5-hydroxy-2 H-benzothiazine-3-carboxylic acid and 7-(3',4'-dihydro-2 H-chromene-3',5',7'-triol)-3,4-dihydro-5-hydroxy-2 H-benzothiazine-3-carboxylic acid) from 2'-cysteinyl catechin and 5'-cysteinyl catechin in cysteine incubations, respectively. In contrast, arginine and lysine did not lead to any amino acid adducts. Target compounds were separated by high-performance countercurrent chromatography and preparative HPLC and unequivocally characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. Mechanistic incubations starting from the catechin-cysteine adducts showed that both catechin and PPO are crucial components in the formation of the dihydrobenzothiazines. The cysteine incubations showed a red-brown coloration, which coincided with formation and degradation of the dihydrobenzothiazines finally leading to the formation of high-polymeric melanins. Therefore, these compounds might be the key intermediates to understand development of color during cysteine-driven enzymatic browning reactions.

Knockout of the S-acyltransferase Gene, PbPAT14, Confers the Dwarf Yellowing Phenotype in First Generation Pear by ABA Accumulation.

The development of dwarf fruit trees with smaller and compact characteristics leads to significantly increased fruit production, which is a major objective of pear (Pyrus bretschneideri) breeding. We identified the S-acylation activity of PbPAT14, an S-acyltransferase gene related to plant development, using a yeast (Saccharomyces cerevisiae) complementation assay, and also PbPAT14 could rescue the growth defect of the Arabidopsis mutant atpat14. We further studied the function of PbPAT14 by designing three guide RNAs for PbPAT14 to use in the CRISPR/Cas9 system. We obtained 22 positive transgenic pear lines via Agrobacterium-mediated transformation using cotyledons from seeds of Pyrus betulifolia ('Duli'). Six of these lines exhibited the dwarf yellowing phenotype and were homozygous mutations according to sequencing analysis. Ultrastructure analysis suggested that this dwarfism was manifested by shorter, thinner stems due to a reduction in cell number. A higher level of endogenous abscisic acid (ABA) and a higher transcript level of the ABA pathway genes in the mutant lines revealed that the PbPAT14 function was related to the ABA pathway. Overall, our experimental results increase the understanding of how PATs function in plants and help elucidate the mechanism of plant dwarfism.

Transcriptomic and evolutionary analyses of white pear (Pyrus bretschneideri) ß-amylase genes reveals their importance for cold and drought stress responses.

ß-amylase (BAM) genes play essential roles in plant abiotic stress responses. Although the genome of Chinese white pear (Pyrus bretschneideri) has recently been made available, knowledge regarding the BAM family in pear, including gene function, evolutionary history and patterns of gene expression remains limited. In this study, we identified 17 PbBAMs in the pear genome. Of these, 12 PbBAM members were mapped onto 9 chromosomes and 5 PbBAM genes were located on scaffold contigs. Based on gene structure, protein motif analysis, and the topology of the phylogenetic tree of the PbBAM family, we classified member genes into 4 groups. All PbBAM genes were found to contain typical glycosyl hydrolysis 14 domain motifs. Interfamilial comparisons revealed that the phylogenetic relationships of BAM genes in other Rosaceae species were similar those found in pear. We also found that whole-genome duplication (WGD)/segmental duplication events played critical roles in the expansion of the BAM family. Next, we used transcriptomic data to study gene expression during the response of drought and low temperate responses, and found that genes in Group B were related to drought and cold stress. We identified four PbBAM genes associated with abiotic stress in Pear. Finally, by analyzing co-expression networks and co-regulatory genes, we found that PbBAM1a and PbBAM1b were associated with the pear abiotic stress response.

Full inhibition of Whangkeumbae pear polyphenol oxidase enzymatic browning reaction by l-cysteine.

The enzymatic browning reaction caused by polyphenol oxidase (PPO) has a negative effect on the processing of fruits and vegetables. However, some chemical inhibitors have been used to prevent enzymatic browning reaction, although they may be toxic and potentially hazardous to use in food. In this study, PPO was isolated and purified from the Whangkeumbae pear, and four food grade inhibitors were used to prevent the enzymatic browning reaction. The results showed that the PPO activity increased by 32.93-fold; its yield was 0.2 U/100 U, and the specific activity was 519,895.73 U/mg protein. The molecular weight of the PPO was approximately 44 kDa. The most potent inhibitor was l-cysteine, which fully inhibited the PPO activity at a concentration of 0.8 mg/mL. The type of inhibition of l-cysteine was noncompetitive. It suggests that l-cysteine can be utilized to prevent enzymatic browning reaction during the processing of pear juice.

Genome-wide identification of lectin receptor kinases in pear: Functional characterization of the L-type LecRLK gene PbLRK138.

Lectin receptor-like kinases (LecRLKs) are membrane-bound receptors that are believed to be involved in biotic and abiotic stress responses. However, little is known about the LecRLK family in pear. In this study, a total of 172 LecRLK genes were first identified in the entire pear genome. The 172 LecRLKs were divided into three types (111 G-, 59 L- and two C-types) based on their structure and phylogenetic relationships. LecRLKs gene expressions were detected in different pear tissues including roots, stems, leaves, flowers and fruits, and the most of the 11 selected LecRLKs exhibited similar expression patterns. Furthermore, six selected LecRLKs were shown to be involved in salt stress response. Overexpression of PbLRK138, an L-type LecRLK, caused cell death and induced expression of defense-related genes in Nicotiana benthamiana. Two deletion mutants containing lectin or transmembrane and serine/threonine kinase domains did not trigger cell death. In addition, only the mutant with the transmembrane domain was localized to the plasma membrane.