A Combined Metabolome and Transcriptome Reveals the Lignin Metabolic Pathway during the Developmental Stages of Peel Coloration in the 'Xinyu' Pear.

Sand pear is the main cultivated pear species in China, and brown peel is a unique feature of sand pear. The formation of brown peel is related to the activity of the cork layer, of which lignin is an important component. The formation of brown peel is intimately associated with the biosynthesis and accumulation of lignin; however, the regulatory mechanism of lignin biosynthesis in pear peel remains unclear. In this study, we used a newly bred sand pear cultivar 'Xinyu' as the material to investigate the biosynthesis and accumulation of lignin at nine developmental stages using metabolomic and transcriptomic methods. Our results showed that the 30 days after flowering (DAF) to 50DAF were the key periods of lignin accumulation according to data analysis from the assays of lignin measurement, scanning electron microscope (SEM) observation, metabolomics, and transcriptomics. Through weighted gene co-expression network analysis (WGCNA), positively correlated modules with lignin were identified. A total of nine difference lignin components were identified and 148 differentially expressed genes (DEGs), including 10 structural genes (PAL1, C4H, two 4CL genes, HCT, CSE, two COMT genes, and two CCR genes) and MYB, NAC, ERF, and TCP transcription factor genes were involved in lignin metabolism. An analysis of RT-qPCR confirmed that these DEGs were involved in the biosynthesis and regulation of lignin. These findings further help us understand the mechanisms of lignin biosynthesis and provide a theoretical basis for peel color control and quality improvement in pear breeding and cultivation.

Effect of 1-MCP and ethylene absorbent on the development of lenticel disorder of 'Xinli No.7' pear and possible mechanisms.

BACKGROUD: A common lenticel disorder which occurs in the peel of 'Xinli No. 7' pears (Pyrus bretschneideri Rehd.) had not previously been described. Symptoms of this lenticel disorder include enlarging and bulging of the lenticels which results in significant commercial losses. Understanding the physiological basis of lenticel disorder and developing practical methods to control it is crucial for the successful marketing of this pear. RESULTS: The development of this lenticel disorder was found to be closely related to the endogenous ethylene production during storage. 1-Methylcyclopropene (1-MCP) combined with an ethylene absorbent (EA) treatment was found to significantly reduce the development of the disorder by inhibiting the expression of ethylene related genes, PbACS1, PbACS2 and PbACO. It is proposed that the enlarged lenticels may result from increased lignin accumulation in the peel cells, which is inhibited by this combined postharvest treatment. It was shown that the expression of six lignin related genes decreased following the treatment. The results suggest that PbPAL, Pb4CL and PbCAD could be critical in regulating the development of this lenticel disorder. CONCLUSION: Endogenous ethylene plays a key role in the development of this lenticel disorder in 'Xinli No. 7' pear. The enlarged lenticels which is characteristic of this disorder maybe related to increased lignin accumulation in the peel cells, which were inhibited with 1-MCP combined with an EA treatment. These results provide a practical method for managing the development of lenticel disorder in 'Xinli No. 7' pear and helps clarify the developmental mechanisms of this disorder. © 2020 Society of Chemical Industry.

Transcriptomic analysis of early fruit development in Chinese white pear (Pyrus bretschneideri Rehd.) and functional identification of PbCCR1 in lignin biosynthesis.

BACKGROUND: The content of stone cells and lignin is one of the key factors affecting the quality of pear fruit. In a previous study, we determined the developmental regularity of stone cells and lignin in 'Dangshan Su' pear fruit 15-145 days after pollination (DAP). However, the development of fruit stone cells and lignin before 15 DAP has not been heavily researched. RESULTS: In this study, we found that primordial stone cells began to appear at 7 DAP and that the fruit had formed a large number of stone cells at 15 DAP. Subsequently, transcriptome sequencing was performed on fruits at 0, 7, and 15 DAP and identified 3834 (0 vs. 7 DAP), 4049 (7 vs. 15 DAP) and 5763 (0 vs. 15 DAP) DEGs. During the 7-15 DAP period, a large number of key enzyme genes essential for lignin biosynthesis are gradually up-regulated, and their expression pattern is consistent with the accumulation of lignin in this period. Further analysis found that the biosynthesis of S-type lignin in 'Dangshan Su' pear does not depend on the catalytic activity of PbSAD but is primarily generated by the catalytic activity of caffeoyl-CoA through CCoAOMT, CCR, F5H, and CAD. We cloned PbCCR1, 2 and analysed their functions in Chinese white pear lignin biosynthesis. PbCCR1 and 2 have a degree of functional redundancy; both demonstrate the ability to participate in lignin biosynthesis. However, PbCCR1 may be the major gene for lignin biosynthesis, while PbCCR2 has little effect on lignin biosynthesis. CONCLUSIONS: Our results revealed that 'Dangshan Su' pear began to form a large number of stone cells and produce lignin after 7 DAP and mainly accumulated materials from 0 to 7 DAP. PbCCR1 is mainly involved in the biosynthesis of lignin in 'Dangshan Su' pear and plays a positive role in lignin biosynthesis.

PbrmiR397a regulates lignification during stone cell development in pear fruit.

Lignified stone cells substantially reduce fruit quality. Therefore, it is desirable to inhibit stone cell development using genetic technologies. However, the molecular mechanisms regulating lignification are poorly understood in fruit stone cells. In this study, we have shown that microRNA (miR) miR397a regulates fruit cell lignification by inhibiting laccase (LAC) genes that encode key lignin biosynthesis enzymes. Transient overexpression of PbrmiR397a, which is the miR397a of Chinese pear (Pyrus bretschneideri), and simultaneous silencing of three LAC genes reduced the lignin content and stone cell number in pear fruit. A single nucleotide polymorphism (SNP) identified in the promoter of the PbrmiR397a gene was found to associate with low levels of fruit lignin, after analysis of the genome sequences of sixty pear varieties. This SNP created a TCA element that responded to salicylic acid to induce gene expression as confirmed using a cell-based assay system. Furthermore, stable overexpression of PbrmiR397a in transgenic tobacco plants reduced the expression of target LAC genes and decreased the content of lignin but did not change the ratio of syringyl- and guaiacyl-lignin monomers. Consistent with reduction in lignin content, the transgenic plants showed fewer numbers of vessel elements and thinner secondary walls in the remaining elements compared to wild-type control plants. This study has advanced our understanding of the regulation of lignin biosynthesis and provided useful molecular genetic information for improving pear fruit quality.

PbrMYB169 positively regulates lignification of stone cells in pear fruit.

Stone cells negatively affect fruit quality because of their firm and lignified cell walls, so are targets for reduction in pear breeding programmes. However, there is only limited knowledge of the molecular mechanisms underlying the formation of stone cells. Here, we show that PbrMYB169, an R2R3 MYB transcription factor, of Pyrus bretschneideri positively regulates lignification of stone cells in pear fruit. PbrMYB169 was shown to be co-expressed with lignin biosynthesis genes during pear fruit development, and this co-expression pattern was coincident with stone cell formation in the fruit of Pyrus bretschneideri 'Dangshansuli'. The PbrMYB169 expression level was also positively correlated with stone cell content in 36 pear cultivars tested. PbrMYB169 protein significantly activated the promoter of lignin genes C3H1, CCR1, CCOMT2, CAD, 4CL1, 4CL2, HCT2, and LAC18 via binding to AC elements [ACC(T/A)ACC] in these promoters. Furthermore, overexpression of PbrMYB169 in transgenic Arabidopsis plants enhanced the expression of lignin genes, and increased lignin deposition and cell wall thickness of vessel elements, but did not change the ratio of syringyl and guaiacyl lignin monomers. In conclusion, PbrMYB169 appears to be a transcriptional activator of lignin biosynthesis and regulates secondary wall formation in fruit stone cells. This study advances the understanding of the regulation of lignin biosynthesis and provides valuable molecular genetic information for reducing stone cell content in pear fruit.

Systematic analysis and comparison of the PHD-Finger gene family in Chinese pear (Pyrus bretschneideri) and its role in fruit development.

PHD-finger proteins, which belongs to the type of zinc finger family, and that play an important role in the regulation of both transcription and the chromatin state in eukaryotes. Currently, PHD-finger proteins have been well studied in animals, while few studies have been carried out on their function in plants. In the present study, 129 non-redundant PHD-finger genes were identified from 5 Rosaceae species (pear, apple, strawberry, mei, and peach); among them, 31 genes were identified in pear. Subsequently, we carried out a bioinformatics analysis of the PHD-finger genes. Thirty-one PbPHD genes were divided into 7 subfamilies based on the phylogenetic analysis, which are consistent with the intron-exon and conserved motif analyses. In addition, we identified five segmental duplication events, implying that the segmental duplications might be a crucial role in the expansion of the PHD-finger gene family in pear. The microsynteny analysis of five Rosaceae species showed that there were independent duplication events in addition to the genome-wide duplication of the pear genome. Subsequently, ten expressed PHD-finger genes of pear fruit were identified using qRT-PCR, and one of these genes, PbPHD10, was identified as an important candidate gene for the regulation of lignin synthesis. Our research provides useful information for the further analysis of the function of PHD-finger gene family in pear.

Identification of miRNAs involved in pear fruit development and quality.

BACKGROUND: MicroRNAs (miRNAs) are a class of small, endogenous RNAs that take part in regulating genes through mediating gene expressions at the post-transcriptional level in plants. Previous studies have reported miRNA identification in various plants ranging from model plants to perennial fruit trees. However, the role of miRNAs in pear (Pyrus bretschneideri) fruit development is not clear. Here, we investigated the miRNA profiles of pear fruits from different time stages during development with Illumina HiSeq 2000 platform and bioinformatics analysis. Quantitative real-time PCR was used to validate the expression levels of miRNAs. RESULTS: Both conserved and species-specific miRNAs in pear have been identified in this study. Total reads, ranging from 19,030,925 to 25,576,773, were obtained from six small RNA libraries constructed for different stages of fruit development after flowering. Comparative profiling showed that an average of 90 miRNAs was expressed with significant differences between various developmental stages. KEGG pathway analysis on 2,216 target genes of 188 known miRNAs and 1,127 target genes of 184 novel miRNAs showed that miRNAs are widely involved in the regulation of fruit development. Among these, a total of eleven miRNAs putatively participate in the pathway of lignin biosynthesis, nine miRNAs were identified to take part in sugar and acid metabolism, and MiR160 was identified to regulate auxin response factor. CONCLUSION: Comparative analysis of miRNAomes during pear fruit development is presented, and miRNAs were proved to be widely involved in the regulation of fruit development and formation of fruit quality, for example through lignin synthesis, sugar and acid metabolism, and hormone signaling. Combined with computational analysis and experimental confirmation, the research contributes valuable information for further functional research of microRNA in fruit development for pear and other species.

PbCSE1 promotes lignification during stone cell development in pear (Pyrus bretschneideri) fruit.

Pear [Pyrus bretschneideri cv. Dangshan Su] fruit quality is not always satisfactory owing to the presence of stone cells, and lignin is the main component of stone cells in pear fruits. Caffeoyl shikimate esterase (CSE) is a key enzyme in the lignin biosynthesis. Although CSE-like genes have been isolated from a variety of plant species, their orthologs are not characterized in pear. In this study, the CSE gene family (PbCSE) from P. bretschneideri was identified. According to the physiological data and quantitative RT-PCR (qRT-PCR), PbCSE1 was associated with lignin deposition and stone cell formation. The overexpression of PbCSE1 increased the lignin content in pear fruits. Relative to wild-type (WT) Arabidopsis, the overexpression of PbCSE1 delayed growth, increased the lignin deposition and lignin content in stems. Simultaneously, the expression of lignin biosynthetic genes were also increased in pear fruits and Arabidopsis. These results demonstrated that PbCSE1 plays an important role in cell lignification and will provide a potential molecular strategy to improve the quality of pear fruits.

Biochemical characterization and expression analysis of lignification in two pear (Pyrus ussuriensis Maxim.) varieties with contrasting stone cell content.

As lignified stone cells reduce fruit quality, we investigated lignin deposition, phenolic metabolites, and expression of lignin biosynthetic genes during fruit development to elucidate the molecular mechanism of stone cell lignification using histological, biochemical, and transcriptional data from two Ussurian pear varieties (Jianba and Nanguo) with contrasting stone cell content. Lignin content and distribution coincided with stone cell accumulation. As per LC-MS analysis, Jianba exhibited higher levels of lignin monomers and hydroxycinnamates than Nanguo, consistently with lignin amount in each case. However, flavonoid content was much higher in Nanguo. Transcriptional data showed that most monolignol biosynthesis-related genes were particularly upregulated in Jianba during lignin accumulation; especially CCR and LAC, two monolignol biosynthesis-specific genes, were substantially upregulated in Jianba fruits at critical stages. Therefore, differences in stone cell content between "Jianba" and "Nanguo" may result from differential expression of lignin synthase genes located downstream of the lignin biosynthesis pathway. Taken together, our data may provide a deeper understanding of the molecular mechanism for stone cell lignification in pear fruit.

Heterogenous expression of Pyrus pyrifolia PpCAD2 and PpEXP2 in tobacco impacts lignin accumulation in transgenic plants.

Lignin, a natural macromolecular compound, plays an important role in the texture and taste of fruit. Hard end is a physiological disorder of pear fruit, in which the level of lignification in fruit tissues is dramatically elevated. Cinnamyl alcohol dehydrogenase and expansin genes (PpCAD2 and PpEXP2, respectively) exhibit higher levels of expression in 'Whangkeumbae' (Pyrus pyrifolia) pear fruit exhibiting this physiological disorder, relative to control fruit without symptoms. These genes were isolated from pear fruit and subsequently expressed in tobacco (Nicotiana tabacum) to investigate their function. Histochemical staining for lignin revealed that the degree of lignification in leaf veins and stem tissues increased in plants transformed with sense constructs and decreased in plants transformed with antisense constructs of PpCAD2. The expression of native NtCADs was also inhibited in the antisense PpCAD2 transgenic tobacco. Sense and antisense PpCAD2 transgenic tobacco exhibited an 86.7% increase and a 60% decrease in CAD activity, respectively, accompanied by a complementary response in lignin content in root tissues. The basal portion of the stem in PpEXP2 transgenic tobacco was bent and highly lignified. Additionally, the level of cellulose also increased in the stem of PpEXP2 transgenic tobacco. Collectively, these results suggested that PpCAD2 and PpEXP2 genes play a significant role in lignin accumulation in transgenic tobacco plants, and it is inferred that these two genes may also participate in the increased lignification observed in hard end pear fruit.

Oxidative stress responses and root lignification induced by Fe deficiency conditions in pear and quince genotypes.

We analysed Pyrus communis cv. Conference and Cydonia oblonga BA29, differently tolerant to lime-induced chlorosis, to identify the key mechanisms involved in their different performance under Fe deficiency induced by the absence of Fe (-Fe) or by the presence of bicarbonate (+FeBic). Under our experimental conditions, a decrease in root elongation was observed in BA29 under bicarbonate supply. Superoxide dismutase (SOD) and peroxidase (POD) activities were analysed and the relative isoforms were detected by native electrophoresis. The data obtained for both genotypes under -Fe and for BA29 +FeBic suggest the occurrence of overproduction of reactive oxygen species (ROS) and, at the same time, of a scarce capacity to detoxify them. The detection of ROS (O(2)(-) and H(2)O(2)) through histochemical localization supports these results and suggests that they could account for the modifications of mechanical properties of the cell wall during stress adaptation. On the other hand, in the cv. Conference +FeBic, an increase in non-specific POD activity was detected, confirming its higher level of protection in particular against H(2)O(2) accumulation. Peroxidases involved in lignification were assayed and histochemical analysis was performed. The results suggest that only in BA29 under bicarbonate supply can the presence of ROS in root apoplast be correlated with lignin deposits in external layers and in endodermis as a consequence of the shift of PODs towards a lignification role. We suggest that in BA29 the decrease in root growth could impair mineral nutrition, generating susceptibility to calcareous soils. In the cv. Conference, the allocation of new biomass to the root system could improve soil exploration and consequently Fe uptake.