Transcription factor PbMYB80 regulates lignification of stone cells and undergoes RING finger protein PbRHY1-mediated degradation in pear fruit.

The Chinese white pear (Pyrus bretschneideri) fruit carries a high proportion of stone cells, adversely affecting fruit quality. Lignin is a main component of stone cells in pear fruit. In this study, we discovered that a pear MYB transcription factor, PbMYB80, binds to the promoters of key lignin biosynthesis genes and inhibits their expression. Stable overexpression of PbMYB80 in Arabidopsis showed that lignin deposition and secondary wall thickening were inhibited, and the expression of the lignin biosynthesis genes in transgenic Arabidopsis was decreased. Transient overexpression of PbMYB80 in pear fruit inhibited lignin metabolism and stone cell development, and the expression of some genes in the lignin metabolism pathway was reduced. In contrast, silencing PbMYB80 with VIGS increased the lignin and stone cell content in pear fruit, and increased expression of genes in the lignin metabolism pathway. By screening a pear fruit cDNA library in yeast, we found that PbMYB80 binds to a RING finger (PbRHY1) protein. We also showed that PbRHY1 exhibits E3 ubiquitin ligase activity and degrades ubiquitinated PbMYB80 in vivo and in vitro. This investigation contributes to a better understanding of the regulation of lignin biosynthesis in pear fruit, and provides a theoretical foundation for increasing pear fruit quality at the molecular level.

Stone cell formation in the pedicel of pears and apples.

MAIN CONCLUSION: For the first time, stone cells in pear and apple pedicel were studied. The lignification of the pedicel outer part was correlated with flesh, and the secondary cell wall biosynthesis genes were activated. Fruit pedicels act as bridges between the fruit and the shoot. They have secondary thickened cell walls that presumably function in mechanical support, water and nutrient transport. Stone cells are cells with a secondary cell wall thickening. In pears, yet not in apples, the stone cells affect the flesh texture. There have been few reports on stone cell formation in pear and apple pedicels; therefore, we studied these cells for the first time. The apple pedicel had few stone cells in the cortex. The formation of stone cells in pear continued until seven weeks after flowering (WAF), and the density was significantly higher than in apple. The stone cell formation degree (SFD) of pear was 3.6-7.1 times higher than that of apple. Total lignin and lignin non-condensed structure (G and S units) content in the pear pedicle outer part was 1.5-2.7 times higher than that of the apple at harvest. The SFD of the pedicel outer part had a positive correlation with the G and S units content of the flesh. The total lignin and G and S units content between flesh and the pedicel outer part were positively correlated. Correlation analysis revealed a positive relationship between fruit and pedicel formation of the stone cells. The WGCNA showed that NST3 was linked to NAC028, MYB46, CESA, POD, LAC, and VSR6. These genes were highly expressed in the outer part of the pear pedicel, while they were suppressed in that issue of the apple at 4 WAF.

Diversity, phylogeny and evolution of the rapidly evolving genus Psidium L. (Myrtaceae, Myrteae).

BACKGROUND AND AIMS: Psidium is the fourthth largest genus of Myrtaceae in the Neotropics. Psidium guajava is widely cultivated in the tropics for its edible fruit. It is commercially under threat due to the disease guava decline. Psidium cattleyanum is one of the 100 most invasive organisms in the world. Knowledge of the phylogenetic relationships within Psidium is poor. We aim to provide a review of the biology, morphology and ecology of Psidium, a phylogenetic tree, an infrageneric classification and a list of species. METHODS: Morphological and geographic data were obtained by studying Psidium in herbaria and in the field between 1988 and 2020. Forty-six herbaria were visited personally. A database of approx. 6000 specimens was constructed, and the literature was reviewed. Thirty species (about a third of the species in the genus) were sampled for molecular phylogenetic inference. Two chloroplast (psbA-trnH and ndhF) and two nuclear (external transcribed spacer and internal transcribed spacer) regions were targeted. Phylogenetic trees were constructed using maximum likelihood (ML; RaxML) and Bayesian inference (BI; MrBayes). KEY RESULTS: Psidium is a monophyletic genus with four major clades recognized as sections. Section Psidium (ten species), to which P. guajava belongs, is sister to the rest of the genus; it is widespread across the Neotropics. Section Obversifolia (six species; restricted to the Brazilian Atlantic Forest), which includes P. cattleyanum, is sister to the innermost clade composed of sister sections Apertiflora (31 species; widespread but most diverse in the Brazilian Atlantic Forest) + Mitranthes (26 species; widespread in dry forests and probably diverse in the Caribbean). Characters associated with diversification within Psidium are discussed. CONCLUSIONS: Research on pre-foliation, colleters, leaf anatomy, leaf physiology, staminal development, placentation and germination associated with the anatomy of the opercular plug is desirable. Studies are biased towards sections Psidium and Obversifolia, with other sections poorly known.

PbUGT72AJ2-Mediated Glycosylation Plays an Important Role in Lignin Formation and Stone Cell Development in Pears (Pyrus bretschneideri).

Glycosylation is necessary for many processes of plant secondary metabolism. It can maintain plant homeostasis and is of great significance to normal plant growth and development. At present, the significance of glycosylation for lignin biosynthesis has been proven in some plants, but it has not yet been reported in pears. We used in situ hybridization, in vitro expression, substrate catalysis, transgenic Arabidopsisthaliana, and transient transformation of pear fruit in our investigation, which was predicated on the identification of a gene PbUGT72AJ2 that may be involved in lignin monolignol glycosylation according to our previous work. These results revealed that PbUGT72AJ2 transcripts were localized to some pulp cell walls, lignin deposition, and stone cell areas of pear fruit. The recombinant PbUGT72AJ2-pGEX4T-1 protein had activity against coniferyl alcohol and sinapyl alcohol, and its catalytic efficiency against coniferyl alcohol was higher than that against sinapyl alcohol. When PbUGT72AJ2 was transferred into Arabidopsisthaliana mutants, it was found that some characteristics of Arabidopsisthalianaugt72e3 mutants were restored. In Arabidopsisthaliana, overexpression of PbUGT72AJ2 enhanced the contents of coniferin and syringin, whereas lignification did not change significantly. Transient transformation of pear fruit showed that when PbUGT72AJ2 in pear fruit was silenced by RNA interference, the content of lignin and stone cells in pear fruit increased, whereas the gene PbUGT72AJ2 was overexpressed in pear fruit, and there was almost no change in the pear fruit compared with the control. Lignin deposition in pear fruit was closely related to stone cell development. In this study, we proved that PbUGT72AJ2 plays an important role in lignin deposition and stone cell development in pear fruit, which provides a molecular biological basis for improving pear fruit quality at the molecular level.

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.

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.

A molecular and genomic reference system for conifer defence against insects.

Insect pests are part of natural forest ecosystems contributing to forest rejuvenation but can also cause ecological disturbance and economic losses that are expected to increase with climate change. The white pine or spruce weevil (Pissodes strobi) is a pest of conifer forests in North America. Weevil-host interactions with various spruce (Picea) species have been explored as a genomic and molecular reference system for conifer defence against insects. Interactions occur in two major phases of the insect life cycle. In the exophase, adult weevils are free-moving and display behaviour of host selection for oviposition that is affected by host traits. In the endophase, insects live within the host where mobility and development from eggs to young adults are affected by a complex system of host defences. Genetic resistance exists in several spruce species and involves synergism of constitutive and induced chemical and physical defences that comprise the conifer defence syndrome. Here, we review conifer defences that disrupt the weevil life cycle and mechanisms by which trees resist weevil attack. We highlight molecular and genomic aspects and a possible role for the weevil microbiome. Knowledge of this conifer defence system is supporting forest health strategies and tree breeding for insect resistance.

Identification of Chinese herbal medicines by fluorescence microscopy: fluorescent characteristics of medicinal bark.

Medicinal bark refers to structures outside the vascular cambium of stems, branches and roots of gymnospermous and dicotyledonous plants that are used as medicinal materials; bark is an important type of Chinese herbal medicine. However, identification of the species from which the bark comes can be very difficult, especially when the bark is dried and sliced. In our previous studies, we have found that fluorescence microscopy is a powerful tool for the identification of easily confused Chinese herbal medicines, powdered Chinese herbal medicines and decoction dregs. To establish the fluorescent characteristics by which medicinal barks can be identified, for ensuring their safe and effective use, a systematic microscopic investigation by normal light and fluorescence microscope was carried out on transverse section samples of 11 medicinal barks commonly used in China. Specifically, the fluorescent characteristics of mechanical tissues, including stone cells and fibres as well as secretory tissues, have been observed. The microscopic features of medicinal bark are here systematically and comparatively described and illustrated. Under the fluorescence microscope, various tissues emitted fluorescence of different colours, and we found that both the colours and the intensity can be used to distinguish and identify these barks.

Analysis of the ß-Glucosidase Family Reveals Genes Involved in the Lignification of Stone Cells in Chinese White Pear (Pyrus bretschneideri Rehd.).

BGLU ß-glucosidases in glycoside hydrolase family 1 (GH1) are involved in many processes of plant secondary metabolism. In particular, its de-glycosylation function plays an important role in the transport of lignin monolignols. No comprehensive study of the BGLU family in Chinese pear (Pyrus bretschneideri Rehd.) has been reported yet. In this study, the 50 BGLU family members from Chinese white pear were identified. Three candidate genes, PbBGLU1, PbBGLU15, and PbBGLU16, that may be involved in lignin synthesis were screened by bioinformatics analysis and qRT-PCR. Subcellular localization showed that all three of these candidate genes were expressed in the extracellular region. Then, we analyzed the functions of PbBGLU1 and PbBGLU16. In situ hybridization analysis showed that PbBGLU1 transcripts were not only localized to some pulp cell walls, lignin deposition, and stone cell areas of a pear fruit, but that was also a small amount of enrichment in normal pear flesh cells. PbBGLU16 transcripts were only enriched in lignin deposition and stone cell areas of pear fruit. Enzyme activity analysis revealed that GST-PbBGLU1 and GST-PbBGLU16 had a stronger activity and higher catalytic efficiency for coniferin than syringin. In addition, GST-PbBGLU16 exhibited the higher activity and catalytic efficiency for the two substrates compared with GST-PbBGLU1. The transformation of PbBGLU1 and PbBGLU16 into Arabidopsis identified that the lignin contents of Arabidopsis BGLU-45 mutant, PbBGLU1-RE, and PbBGLU16-RE were not changed than that of wild-type. However, compared with wild-type Arabidopsis, the overexpression of the plant's lignin increased in varying degrees. The effect of PbBGLU16 on the lignin increment was greater than that of PbBGLU1 in Arabidopsis. In pear fruits, with transient overexpression of PbBGLU1, the contents of lignin and stone cells were significantly higher (0.01 < P < 0.05) than those with empty vector injection pear fruits. After transient expression of PbBGLU16, lignin in pear fruit increased significantly (0.01 < P < 0.05) and stone cells showed a very significant difference (P < 0.01) compared with the control group. However, RNA interference silenced these two genes in pear fruit, which seemed to have no impression on lignin and stone cells. This study provides a molecular biological basis for improving pear fruit quality at the molecular level.

Effects of freeze-thaw cycles on the texture of Nanguo pear.

Freezing is a way to preserve the quality of fruit for a long time. Nanguo pear stored at low temperature is prone to browning and lignification. In this study, freeze-thaw cycles were used to simulate the temperature fluctuation in the process, storage, and transportation. The texture properties were taken as the research focus to analyze the lignification phenomenon of Nanguo pear under freeze-thaw cycles. The results showed that freeze-thaw treatment significantly reduced the firmness and propectin content of Nanguo pear, increased the content of stone cells in the fruit, but also destroyed the size of stone cells in the fruit. However, with the increase of freezing-thawing cycles, the content of lignin, stone cell content, and PAL activity increased significantly, while the content of chlorogenic acid increased first and then decreased. These results are helpful to further understand the correlation between texture change with fruit firmness and formation mechanism of stone cells during freeze-thaw cycles of Nanguo pear.

Morphological and Developmental Features of Stone Cells in Eriobotrya Fruits.

Some members of the Rosaceae family, particularly pear, contain stone cells in their fruits. Although stone cells in pear fruits are well studied, relatively little attention has been given to loquat stone cells. Only a few reports have suggested a relationship between stone cell traits and storage and transport tolerance of loquat fruits. Previously, we generated the variety JT8 from the interspecific hybrid of the loquat cultivar Jiefangzhong (JFZ; Eriobotrya japonica Lindl. cv. Jiefangzhong, female parent) and wild Taiwanese loquat (TL; E. deflexa Nakai, male parent). The JT8 fruits had a granular feel, similar to that of pear fruits, due to the presence of stone cells. In this study, the shape, size, development, and distribution dynamics of stone cells of Eriobotrya plants were thoroughly investigated. The results showed that loquat stone cells are brachysclereids and often contain typical branching pits. Loquat stone cells were distributed as both single stone cells and in stone cell clusters (SCCs), and the density of the stone cells near the core was higher than that near the peel. Stone cell density first increased and then decreased during fruit development. These traits noted in Eriobotrya were very similar to those observed in pear, indicating a close relationship between loquat and pear. Moreover, the contents, density dynamics, and aggregation traits of stone cells of the interspecific hybrid JT8 were derived from the male parent (TL). Transgressive segregation was likely exhibited in the content of stone cells and the size of the SCCs. More specifically, the content of stone cells reached 1.61% (w/w). In extreme cases, SCCs of JT8 exceeded 1,000 µm in length and 500 µm in width. This demonstrated that stone cell traits could be transmitted from parent to progeny through interspecific hybridization. The density dynamics of stone cells in two loquat cultivars with different storage and transport tolerances were also investigated, which indicated that the cultivar with more stone cells was more tolerant to storage and transport. We suggest that wild loquat genetic resources containing stone cells in Eriobotrya plants can be used to gradually improve the storage and transport tolerance of loquat fruits.

Analysis of PRX Gene Family and Its Function on Cell Lignification in Pears (Pyrus bretschneideri).

Class III peroxidases (PRXs) are plant-specific enzymes that play key roles in the responses to biotic and abiotic stress during plant growth and development. In addition, some peroxidases also play roles in plant lignification. In this study, a total of 114 PRX (designated PbPRXs) genes were identified in the pear (Pyrus bretschneideri Rehd) genome based on systematic analysis. These PRX genes were divided into 12 groups based on their phylogenetic relationships. We performed systematic bioinformatics analysis of the PRX genes, including analysis of gene structures, conserved motifs, phylogenetic relationships, and gene expression patterns during pear fruit growth. The PbPRXs are unevenly distributed on the 17 pear chromosomes and some of them on other scaffolds. Gene duplication event analysis indicated that whole-genome duplication (WGD) and segmental duplication play key roles in PRX gene amplification. Ka/Ks analysis suggested that most duplicated PbPRXs experienced purifying selection, with limited functional divergence during the duplication events. Furthermore, the analysis indicated that those highly expressed genes might play significant roles in the lignification of cells to form stone cells in pear fruit. We examined the expression of those highly expressed genes during fruit growth using quantitative real-time PCR (qRT-PCR), verifying differential expression patterns at different stages of fruit. This study provides useful information for further functional analysis of the PRX gene family in pears.

Analysis of Fruit Lignin Content, Composition, and Linkage Types in Pear Cultivars and Related Species.

Lignin content, composition, and linkage types were investigated in pear fruit cultivars and related species. Lignin content increased during early stages and then decreased toward ripening in the core and flesh of "Gold Nijisseiki" and "Alexandrine Douillard". The lignin content was highest at harvest in Chinese quince. Only trace amounts of lignin were detected in apple flesh. The lignin content was low in Japanese pears "Ohshu", "Hosui", and "Kosui", and the noncondensed lignin index was high in flesh. The lignin type was guaiacyl-syringyl (GS) in these pears and related species. The S/G ratio at harvest varied widely (0.75-2.64) and increased during early stages and remained constant toward harvest in "Gold Nijisseiki" and "Alexandrine Douillard". "Gold Nijisseiki" and "Alexandrine Douillard" were determined to be G- and S-lignin-rich types, respectively. ß-Aryl ether, phenylcoumaran, and resinol interunit linkage types were detected among monolignol bonds, and ß-Aryl ether units were the main linkages in the pear.

Molecular identification, phylogenomic characterization and expression patterns analysis of the LIM (LIN-11, Isl1 and MEC-3 domains) gene family in pear (Pyrus bretschneideri) reveal its potential role in lignin metabolism.

Lignin is the main component of stone cells, which are a key factor in determining pear quality. Therefore, modification of lignin biosynthesis has important implications for regulating stone cell formation. LIMs are involved in plant development, stress response and metabolism. However, there is still a lack of knowledge about the pear LIM family and lignin-related LIMs. To address this problem, we identified 14 LIMs from the pear genome and named them. Phylogenomic and feature domain analysis showed that they were divided into CRP- and DA&DAR-LIM groups and five subclades. LIMs from the genomes of four rosids (Prunus mummer, Prunus persica, Fragaria vesca and Vitis vinifera) were also identified, and microsynteny analysis revealed the most orthologous gene pairs in the cross of pear/grape and pear/mei. The transcript levels of PbLIMs were significantly affected by SA, ABA and MeJA. Spatio-temporal expression analysis showed that PbLIMs of the δLIM2 subfamily were highly expressed in the flowers. Changes in the expression levels of PbWLIM1a and PbWLIM1b during fruit development was consistent with the changes in lignin content. Combining phylogenetic analyses, protein three-dimensional structure determination and sequence alignment analyses, these two genes were suggested as lignin-related PbLIMs. Subcellular localization results showed that PbWLIM1a and PbWLIM1b were located mainly in the chloroplast. This study lays the foundation for revealing the mechanism of LIM-mediated lignin metabolism to regulate stone cell formation.

Pharmacognostical evaluation of Psoralea corylifolia Linn. seed.

Psoralea corylifolia Linn. belonging to Fabaceae family is an important endangered plant that has been therapeutically used to treat different pathological manifestations since ages. It is commonly known as Bakuchi in Sanskrit. Though it is an important plant, till date, no pharmacognostical reports have been available on its seed. A lot of adulterations are also present in the market. The present study is aimed towards evaluating pharmacognostical and histochemical characteristics of the seeds of P.corylifolia Linn. in detail. Macroscopic and microscopic pharmacognostical characters of seeds and histochemical studies were noted by following standard methods. Pharmacognostical evaluation of seed shows the presence of volatile oil, silica deposits and stone cells. The observations found in current work can be considered as reference standards in future studies.

Characterization and analysis of CCR and CAD gene families at the whole-genome level for lignin synthesis of stone cells in pear (Pyrus bretschneideri) fruit.

The content of stone cells has significant effects on the flavour and quality of pear fruit. Previous research suggested that lignin deposition is closely related to stone cell formation. In the lignin biosynthetic pathway, cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD), dehydrogenase/reductase family members, catalyse the last two steps in monolignol synthesis. However, there is little knowledge of the characteristics of the CCR and CAD families in pear and their involvement in lignin synthesis of stone cells. In this study, 31 CCRs and 26 CADs were identified in the pear genome. Phylogenetic trees for CCRs and CADs were constructed; key amino acid residues were analysed, and three-dimensional structures were predicted. Using quantitative real-time polymerase chain reaction (qRT-PCR), PbCAD2, PbCCR1, -2 and -3 were identified as participating in lignin synthesis of stone cells in pear fruit. Subcellular localization analysis showed that the expressed proteins (PbCAD2, PbCCR1, -2 and -3) are found in the cytoplasm or at the cell membrane. These results reveal the evolutionary features of the CCR and CAD families in pear as well as the genes responsible for regulation of lignin synthesis and stone cell development in pear fruit.

Identification of powdered Chinese herbal medicines by fluorescence microscopy, Part 1: Fluorescent characteristics of mechanical tissues, conducting tissues, and ergastic substances.

The light microscope has been successfully used in identification of Chinese herbal medicines (CHMs) for more than a century. However, positive identification is not always possible. Given the popularity of fluorescence microscopy in bioanalysis, researchers dedicated to finding new ways to identify CHMs more effectively are now turning to fluorescence microscopy for authentication purposes. Some studies on distinguishing confused species from the same genus and on exploring distributions of chemicals in tissues of CHMs by fluorescence microscopy have been reported; however, no systematic investigations on fluorescent characteristics of powdered CHMs have been reported. Here, 46 samples of 16 CHMs were investigated. Specifically, the mechanical tissues including stone cells and fibers, the conducting tissues including three types of vessels, and ergastic substances including crystals of calcium oxalate and secretions, in various powdered CHMs were investigated by both light microscope and fluorescence microscope. The results showed many microscopic features emit fluorescence that makes them easily observed, even against complex backgrounds. Under the fluorescence microscope, different microscopic features from the same powdered CHM or some same features from different powdered CHMs emitted the different fluorescence, making this information very helpful for the authentication of CHMs in powder form. Moreover, secretions with unique chemical profiles from different powdered CHMs showed different fluorescent characteristics. Hence, fluorescence microscopy could be a useful additional method for the authentication of powdered CHMs if the fluorescent characteristics of specific CHMs are known.