Transcription factor PbNAC71 regulates xylem and vessel development to control plant height.

Dwarfism is an important agronomic trait in fruit breeding programs. However, the germplasm resources required to generate dwarf pear (Pyrus spp.) varieties are limited. Moreover, the mechanisms underlying dwarfism remain unclear. In this study, "Yunnan" quince (Cydonia oblonga Mill.) had a dwarfing effect on "Zaosu" pear. Additionally, the dwarfism-related NAC transcription factor gene PbNAC71 was isolated from pear trees comprising "Zaosu" (scion) grafted onto "Yunnan" quince (rootstock). Transgenic Nicotiana benthamiana and pear OHF-333 (Pyrus communis) plants overexpressing PbNAC71 exhibited dwarfism, with a substantially smaller xylem and vessel area relative to the wild-type controls. Yeast one-hybrid, dual-luciferase, chromatin immunoprecipitation-qPCR, and electrophoretic mobility shift assays indicated that PbNAC71 downregulates PbWalls are thin 1 expression by binding to NAC-binding elements in its promoter. Yeast two-hybrid assays showed that PbNAC71 interacts with the E3 ubiquitin ligase PbRING finger protein 217 (PbRNF217). Furthermore, PbRNF217 promotes the ubiquitin-mediated degradation of PbNAC71 by the 26S proteasome, thereby regulating plant height as well as xylem and vessel development. Our findings reveal a mechanism underlying pear dwarfism and expand our understanding of the molecular basis of dwarfism in woody plants.

PbBPC4 involved in a xylem-deficient dwarf phenotype in pear by directly regulating the expression of PbXND1.

Dwarfing is an important agronomic trait in fruit breeding. At present, dwarf cultivars or dwarfing rootstocks are used for high-density planting. Although some dwarf rootstocks have been used in the cultivation of pear (Pyrus bretschneideri Rehd), the breeding of dwarf pear rootstocks or cultivars is still sorely lacking. A previous study reported that PbXND1 results in a xylem-dwarf phenotype in pear trees. However, the regulatory mechanism upstream of PbXND1 is unclear. In this study, we identified PbBPC4 as an upstream regulatory factor of PbXND1 in yeast one-hybrid assays. In ß-glucuronidase staining and dual-luciferase assays, PbBPC4 enhanced the activity of the PbXND1 promoter. Tobacco plants overexpressing PbBPC4 showed decreased plant height because of a reduced xylem size. Similar changes in the xylem was observed in transgenic pear roots; those overexpressing PbBPC4 showed reduced xylem size, and those with silencing PbBPC4 expression showed increased xylem size, greater density of xylem vessels, and a larger proportion of the xylem out of the total cross-section area. Expression analyses showed that PbBPC4 increases the transcription of PbXND1, leading to reduced transcript levels of genes involved in the positive regulation of xylem development, ultimately resulting in a xylem-deficient dwarf phenotype. Taken together, our results reveal the mechanism by which PbBPC4 participates in the regulation of xylem development via directly altering the expression of PbXND1, thus leading to the dwarf phenotype in pear. These findings have reference value for the breeding of dwarf pear trees.

PbWRKY26 positively regulates malate accumulation in pear fruit by activating PbMDH3.

Malate is the main organic acid that affects fruit acidity and flavor in pear (Pyrus spp.). However, the regulatory mechanism of malic acid accumulation in pear remains unclear. We identified PbWRKY26 as a candidate gene using mRNA-seq, and quantification analysis verified the expression level. The expression of PbWRKY26 was positively correlated with the malic acid content in two P. pyrifolia cultivars ('Cuiguan', 'Hongsucui') and two P. ussuriensis cultivars ('Qiuxiang', 'Hanhong'), with respective correlation coefficients of 0.748*, 0.871**, 0.889**, and 0.910** (*, P < 0.05; **, P < 0.01). The expression of PbWRKY26 enhanced the malate content in overexpression transgenic pear fruit and callus. In contrast, silencing PbWRKY26 decreased the pear fruit malic acid content. Analysis of the neighbor-joining phylogenetic tree indicated that PbWRKY26 was a PH3 homolog. The WRKY26 (PH3) has been identified to regulate a proton pump gene, PH5, in a lot of plant species, but the LUC and Y1H assays showed that PbWRKY26 could not bind to PbPH5 promoter in our study. Interestingly, a malate dehydrogenase gene, PbMDH3, was identified to be regulated by PbWRKY26. This study might be valuable to understand the metabolic regulatory network associated with malate accumulation.

'Yunnan' quince rootstock promoted flower bud formation of 'Abbé Fetel' pear by altering hormone levels and PbAGL9 expression.

Flower busd formation is an important plant growth process. It has been reported that dwarfing rootstocks can significantly affect the flower bud formation of scions. In this study, we found the dwarfing rootstock 'Yunnan' quince could significantly increase the flowering rate of 'Abbé Fetel' pear scions. The RNA-sequencing data revealed significant changes in the expression of genes related to hormone pathways. Furthermore, hormone analyses indicated that 'Yunnan' quince significantly decreased the GA3 content and increased the cytokinin/auxin ratio in 'Abbé Fetel' pear apical buds. The hormone contents were consistent with the RNA-sequencing data. Moreover, we found the flower development-related genes PbAGL9 and PbCAL-A1 were significantly upregulated and PbTFL1 was significantly downregulated in 'Abbé Fetel'/'Yunnan' quince apical buds. To further clarify the relationship between hormones and flowering-related genes, a hormone response assay was carried out. We found the expression levels of PbCAl-A1, PbTFL1 and PbAGL9 were regulated by hormones including GA3, CPPU and NAA. Y1H and dual-luciferase assays indicated that PbAGL9 significantly decreased the promoter activity of PbTFL1. In summary, 'Yunnan' quince upregulated PbCAL-A1 and PbAGL9, and downregulated PbTFL1 expression by decreasing the GA3 content and increasing the cytokinin/auxin ratio in 'Abbé Fetel' pear apical buds. Additionally, 'Yunnan' quince down-regulate PbTFL1 by upregulating the expression of PbAGL9, and eventually promoted floral induction in 'Abbé Fetel' pear.

PbXND1 Results in a Xylem-Deficient Dwarf Phenotype through Interaction with PbTCP4 in Pear (Pyrus bretschneideri Rehd.).

Dwarfing is an important agronomic characteristic in fruit breeding. However, due to the lack of dwarf cultivars and dwarf stocks, the dwarfing mechanism is poorly understood in pears. In this research, we discovered that the dwarf hybrid seedlings of pear (Pyrus bretschneideri Rehd.), 'Red Zaosu,' exhibited a xylem-deficient dwarf phenotype. The expression level of PbXND1, a suppressor of xylem development, was markedly enhanced in dwarf hybrid seedlings and its overexpression in pear results in a xylem-deficient dwarf phenotype. To further dissect the mechanism of PbXND1, PbTCP4 was isolated as a PbXND1 interaction protein through the pear yeast library. Root transformation experiments showed that PbTCP4 promotes root xylem development. Dual-luciferase assays showed that PbXND1 interactions with PbTCP4 suppressed the function of PbTCP4. PbXND1 expression resulted in a small amount of PbTCP4 sequestration in the cytoplasm and thereby prevented it from activating the gene expression, as assessed by bimolecular fluorescence complementation and co-location analyses. Additionally, PbXND1 affected the DNA-binding ability of PbTCP4, as determined by utilizing an electrophoretic mobility shift assay. These results suggest that PbXND1 regulates the function of PbTCP4 principally by affecting the DNA-binding ability of PbTCP4, whereas the cytoplasmic sequestration of PbTCP4 is only a minor factor. Taken together, this study provides new theoretical support for the extreme dwarfism associated with the absence of xylem caused by PbXND1, and it has significant reference value for the breeding of dwarf varieties and dwarf rootstocks of the pear.

Nondestructive Testing of Pear Based on Fourier Near-Infrared Spectroscopy.

Fourier transform near-infrared (FT-NIR) spectroscopy is a nondestructive, rapid, real-time analysis of technical detection methods with an important reference value for producers and consumers. In this study, the feasibility of using FT-NIR spectroscopy for the rapid quantitative analysis and qualitative analysis of 'Zaosu' and 'Dangshansuli' pears is explored. The quantitative model was established by partial least squares (PLS) regression combined with cross-validation based on the spectral data of 340 pear fresh fruits and synchronized with the reference values determined by conventional assays. Furthermore, NIR spectroscopy combined with cluster analysis was used to identify varieties of 'Zaosu' and 'Dangshansuli'. As a result, the model developed using FT-NIR spectroscopy gave the best results for the prediction models of soluble solid content (SSC) and titratable acidity (TA) of 'Dangshansuli' (residual prediction deviation, RPD: 3.272 and 2.239), which were better than those developed for 'Zaosu' SSC and TA modeling (RPD: 1.407 and 1.471). The results also showed that the variety identification of 'Zaosu' and 'Dangshansuli' could be carried out based on FT-NIR spectroscopy, and the discrimination accuracy was 100%. Overall, FT-NIR spectroscopy is a good tool for rapid and nondestructive analysis of the internal quality and variety identification of fresh pears.

PbLAC4-like, activated by PbMYB26, related to the degradation of anthocyanin during color fading in pear.

BACKGROUND: Decrease in anthocyanin content results in the loss of red color in leaves, petals and receptacles during development. The content of anthocyanin was affected by the biosynthesis and degradation of anthocyanin. Compared with the known detailed mechanism of anthocyanin biosynthesis, the degradation mechanism is not fully investigated. It is vital to study the degradation mechanism of anthocyanin in pear for promoting the accumulation of anthocyanin and inhibiting the red fading in pear. RESULTS: Here, we reported that laccase encoded by PbLAC4-like was associated with anthocyanin degradation in pear. The expression pattern of PbLAC4-like was negatively correlated with the content of anthocyanin during the color fading process of pear leaves, petals and receptacles. Phylogenetic analysis and sequence alignment revealed that PbLAC4-like played a vital role in anthocyanin degradation. Thus, the degradation of anthocyanin induced by PbLAC4-like was further verified by transient assays and prokaryotic expression. More than 80% of anthocyanin compounds were degraded by transiently over-expressed PbLAC4-like in pear fruitlet peel. The activity of crude enzyme to degrade anthocyanin in leaves at different stages was basically consistent with the expression of PbLAC4-like. The anthocyanin degradation ability of prokaryotic induced PbLAC4-like protein was also verified by enzyme activity assay. Besides, we also identified PbMYB26 as a positive regulator of PbLAC4-like. Yeast one-hybrid and dual luciferase assay results showed that PbMYB26 activated PbLAC4-like expression by directly binding to the PbLAC4-like promoter. CONCLUSIONS: Taken together, the PbLAC4-like activated by PbMYB26, was involved in the degradation of anthocyanin, resulting in the redness fading in different pear tissues.

Jasmonic Acid and Ethylene Participate in the Gibberellin-Induced Ovule Programmed Cell Death Process in Seedless Pear '1913' (Pyrus hybrid).

Seedless fruit is a feature appreciated by consumers. The ovule abortion process is highly orchestrated and controlled by numerous environmental and endogenous signals. However, the mechanisms underlying ovule abortion in pear remain obscure. Here, we found that gibberellins (GAs) have diverse functions during ovules development between seedless pear '1913' and seeded pear, and that GA4+7 activates a potential programmed cell death process in '1913' ovules. After hormone analyses, strong correlations were determined among jasmonic acid (JA), ethylene and salicylic acid (SA) in seedless and seeded cultivars, and GA4+7 treatments altered the hormone accumulation levels in ovules, resulting in significant correlations between GA and both JA and ethylene. Additionally, SA contributed to ovule abortion in '1913'. Exogenously supplying JA, SA or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid promoted 'Bartlett' seed death. The regulatory mechanism in which ethylene controls ovule death has been demonstrated; therefore, JA's role in regulating '1913' ovule abortion was investigated. A further study identified that the JA signaling receptor MYC2 bound the SENESCENCE-ASSOCIATED 39 promoter and triggered its expression to regulate ovule abortion. Thus, we established ovule abortion-related relationships between GA and the hormones JA, ethylene and SA, and we determined their synergistic functions in regulating ovule death.

PbWRKY75 promotes anthocyanin synthesis by activating PbDFR, PbUFGT, and PbMYB10b in pear.

Anthocyanins are common secondary metabolites in plants that impart red coloration to fruits and flowers. The important WRKY transcription factor family plays multifaceted roles in plant growth and development. In this study, we found a WRKY family gene, Pyrus bretschneideri WRKY75, that may be involved in anthocyanin synthesis in pear. Unlike Arabidopsis thaliana WRKY75, PbWRKY75 may be a positive regulator of anthocyanin synthesis. A transient expression assay indicated that PbWRKY75 promoted pear anthocyanin synthesis. The structural genes (PbANS, PbDFR, and PbUFGT) and positive regulators (PbMYB10 and PbMYB10b) of anthocyanin synthesis were significantly upregulated in the fruitlet skins of PbWRKY75-overexpressing "Zaosu" pears. Subsequently, yeast one-hybrid and dual-luciferase assays indicated that PbWRKY75 promoted PbDFR, PbUFGT, and PbMYB10b expression by activating their promoters. These results revealed that PbWRKY75 may promote the expression of both PbMYB10b and anthocyanin late biosynthetic genes (PbDFR and PbUFGT) by activating their promoters, thereby inducing anthocyanin synthesis in pear. This study enhanced our understanding of the mechanism of pear anthocyanin synthesis, which will be beneficial in the improvement of pear peel color.

Downstream of GA4, PbCYP78A6 participates in regulating cell cycle-related genes and parthenogenesis in pear (Pyrus bretshneideri Rehd.).

BACKGROUND: Parthenocarpy results in traits attractive to both consumers and breeders, and it overcomes the obstacle of self-incompatibility in the fruit set of horticultural crops, including pear (Pyrus bretshneider). However, there is limited knowledge regarding the genetic and molecular mechanisms that regulate parthenogenesis. RESULTS: Here, in a transcriptional comparison between pollination-dependent fruit and GA4-induced parthenocarpy, PbCYP78A6 was identified and proposed as a candidate gene involved in parthenocarpy. PbCYP78A6 is similar to Arabidopsis thaliana CYP78A6 and highly expressed in pear hypanthia. The increased PbCYP78A6 expression, as assessed by RT-qPCR, was induced by pollination and GA4 exposure. The ectopic overexpression of PbCYP78A6 contributed to parthenocarpic fruit production in tomato. The PbCYP78A6 expression coincided with fertilized and parthenocarpic fruitlets development and the expression of fruit development-related genes as assessed by cytological observations and RT-qPCR, respectively. PbCYP78A6 RNA interference and overexpression in pear calli revealed that the gene is an upstream regulator of specific fruit development-related genes in pear. CONCLUSIONS: Our findings indicate that PbCYP78A6 plays a critical role in fruit formation and provide insights into controlling parthenocarpy.

The MIR-Domain of PbbHLH2 Is Involved in Regulation of the Anthocyanin Biosynthetic Pathway in "Red Zaosu" (PyrusBretschneideri Rehd.) Pear Fruit.

The N-terminal of Myc-like basic helix-loop-helix transcription factors (bHLH TFs) contains an interaction domain, namely the MYB-interacting region (MIR), which interacts with the R2R3-MYB proteins to regulate genes involved in the anthocyanin biosynthetic pathway. However, the functions of MIR-domain bHLHs in this pathway are not fully understood. In this study, PbbHLH2 containing the MIR-domain was identified and its function investigated. The overexpression of PbbHLH2 in "Zaosu" pear peel increased the anthocyanin content and the expression levels of late biosynthetic genes. Bimolecular fluorescence complementation showed that PbbHLH2 interacted with R2R3-MYB TFs PbMYB9, 10, and 10b in onion epidermal cells and confirmed that MIR-domain plays important roles in the interaction between the MIR-domain bHLH and R2R3-MYB TFs. Moreover, PbbHLH2 bound and activated the dihydroflavonol reductase promoter in yeast one-hybrid (Y1H) and dual-luciferase assays. Taken together these results suggested that the MIR domain of PbbHLH2 regulated anthocyanin biosynthesis in pear fruit peel.

PbEIL1 acts upstream of PbCysp1 to regulate ovule senescence in seedless pear.

Numerous environmental and endogenous signals control the highly orchestrated and intricate process of plant senescence. Ethylene, a well-known inducer of senescence, has long been considered a key endogenous regulator of leaf and flower senescence, but the molecular mechanism of ethylene-induced ovule senescence has not yet been elucidated. In this study, we found that blockage of fertilization caused ovule abortion in the pear cultivar '1913'. According to transcriptome and phytohormone content data, ethylene biosynthesis was activated by pollination. At the same time, ethylene overaccumulated in ovules, where cells were sensitive to ethylene signals in the absence of fertilization. We identified a transcription factor in the ethylene signal response, ethylene-insensitive 3-like (EIL1), as a likely participant in ovule senescence. Overexpression of PbEIL1 in tomato caused precocious onset of ovule senescence. We further found that EIL1 could directly bind to the promoter of the SENESCENCE-ASSOCIATED CYSTEINE PROTEINASE 1 (PbCysp1) gene and act upstream of senescence. Yeast one-hybrid and dual-luciferase assays revealed the interaction of the transcription factor and the promoter DNA sequence and demonstrated that PbEIL1 enhanced the action of PbCysp1. Collectively, our results provide new insights into how ethylene promotes the progression of unfertilized ovule senescence.

PbGA20ox2 Regulates Fruit Set and Induces Parthenocarpy by Enhancing GA4 Content.

Fruit set and development occur following successful fertilization. Parthenocarpy, a valuable trait in some self-incompatible species, produces seedless fruit without fertilization. Gibberellin (GA) is a crucial hormone in fruit-set regulation and development. While investigating the development of parthenocarpy in pear (Pyrus bretschneideri Rehd.), we determined that GA 20-oxidases (GA20ox) may play key roles in seedless pear fruit development. Sequence analysis revealed three PbGA20ox genes: PbGA20ox1, PbGA20ox2, and PbGA20ox3. We analyzed the expression patterns of candidate genes and found that PbGA20ox2 levels significantly changed in pollinated fruits. Tissue-specific expression assays revealed that PbGA20ox2 is highly expressed in young fruit and leaves. Subcellular localization assays showed it was located in the cytoplasm, nucleus, and plasma membrane. Overexpressed PbGA20ox2 tomato plants were taller and had longer hypocotyls and internodes, and the emasculated flowers produced parthenocarpic fruit. In pear, the transient overexpression of PbGA20ox2 promoted fruit development and delayed the drop of nonpollinated fruit. Furthermore, the fruit of PbGA20ox2-overexpressing tomato and transient PbGA20ox2-overexpressing pear had increased GA4 (but not GA3 and GA1) contents. This result provided evidence that PbGA20ox2 was necessary for GA4-dependent pear fruit development. Our study revealed that PbGA20ox2 altered the GA biosynthetic pathway and enhanced GA4 synthesis, thereby promoting fruit set and parthenocarpic fruit development.

REVEILLE Transcription Factors Contribute to the Nighttime Accumulation of Anthocyanins in 'Red Zaosu' (Pyrus bretschneideri Rehd.) Pear Fruit Skin.

Anthocyanin biosynthesis exhibits a rhythmic oscillation pattern in some plants. To investigate the correlation between the oscillatory regulatory network and anthocyanin biosynthesis in pear, the anthocyanin accumulation and the expression patterns of anthocyanin late biosynthetic genes (ALBGs) were investigated in fruit skin of 'Red Zaosu' (Pyrus bretschneideri Rehd.). The anthocyanin accumulated mainly during the night over three continuous days in the fruit skin, and the ALBGs' expression patterns in 'Red Zaosu' fruit skin were oscillatory. However, the expression levels of typical anthocyanin-related transcription factors did not follow this pattern. Here, we found that the expression patterns of four PbREVEILLEs (PbRVEs), members of a class of atypical anthocyanin-regulated MYBs, were consistent with those of ALBGs in 'Red Zaosu' fruit skin over three continuous days. Additionally, transient expression assays indicated that the four PbRVEs promoted anthocyanin biosynthesis by regulating the expression of the anthocyanin biosynthetic genes encoding dihydroflavonol-4-reductase (DFR) and anthocyanidin synthase (ANS) in red pear fruit skin, which was verified using a dual-luciferase reporter assay. Moreover, a yeast one-hybrid assay indicated that PbRVE1a, 1b and 7 directly bound to PbDFR and PbANS promoters. Thus, PbRVEs promote anthocyanin accumulation at night by up-regulating the expression levels of PbDFR and PbANS in 'Red Zaosu' fruit skin.

PbMYB120 Negatively Regulates Anthocyanin Accumulation in Pear.

Subgroup 4 R2R3 MYBs play vital roles in the regulation of anthocyanin biosynthesis. However, there is limited knowledge regarding the functions of MYB repressors in pear (Pyrus × bretschneideri). Here, PbMYB120 was identified as a potential regulator of anthocyanin biosynthesis. A phylogenetic analysis revealed that PbMYB120 was clustered into the FaMYB1-like clade of the subgroup 4 R2R3 MYBs. PbMYB120 was expressed higher in red peels than in green peels in five pear cultivars. PbMYB120 expression was positively correlated with anthocyanin accumulation. However, the transient overexpression of PbMYB120 led to the inhibition of anthocyanin accumulation and PbUFGT1 expression. Promoter binding and activation assays indicated that PbMYB120 binds to the promoter of PbUFGT1 and represses the promoter's activity. Thus, the inhibition of anthocyanin accumulation by PbMYB120 may be correlated with the repression of PbUFGT1. Furthermore, during anthocyanin induction, the expression levels of anthocyanin activators and PbMYB120 were upregulated. This study demonstrated that PbMYB120 was highly expressed in pear tissues having higher anthocyanin accumulations but acted as a repressor in the regulation of anthocyanin accumulation. PbMYB120 may work coordinately with anthocyanin activators and serve as a balancer of anthocyanin accumulation.

Jasmonate and Ethylene-Regulated Ethylene Response Factor 22 Promotes Lanolin-Induced Anthocyanin Biosynthesis in 'Zaosu' Pear (Pyrusbretschneideri Rehd.) Fruit.

Anthocyanin contributes to the coloration of pear fruit and enhances plant defenses. Members of the ethylene response factor (ERF) family play vital roles in hormone and stress signaling and are involved in anthocyanin biosynthesis. Here, PbERF22 was identified from the lanolin-induced red fruit of 'Zaosu' pear (Pyrus bretschneideri Rehd.) using a comparative transcriptome analysis. Its expression level was up- and down-regulated by methyl jasmonate and 1-methylcyclopropene plus lanolin treatments, respectively, which indicated that PbERF22 responded to the jasmonate- and ethylene-signaling pathways. In addition, transiently overexpressed PbERF22 induced anthocyanin biosynthesis in 'Zaosu' fruit, and a quantitative PCR analysis further confirmed that PbERF22 facilitated the expression of anthocyanin biosynthetic structural and regulatory genes. Moreover, a dual luciferase assay showed that PbERF22 enhanced the activation effects of PbMYB10 and PbMYB10b on the PbUFGT promoter. Therefore, PbERF22 responses to jasmonate and ethylene signals and regulates anthocyanin biosynthesis. This provides a new perspective on the correlation between jasmonate-ethylene crosstalk and anthocyanin biosynthesis.

PbGA2ox8 induces vascular-related anthocyanin accumulation and contributes to red stripe formation on pear fruit.

Fruit with stripes, which are generally longitudinal, can occur naturally, but the bioprocesses underlying this phenomenon are unclear. Previously, we observed an atypical anthocyanin distribution that caused red-striped fruit on the spontaneous pear bud sport "Red Zaosu" (Pyrus bretschneideri Rehd.). In this study, comparative transcriptome analysis of the sport and wild-type "Zaosu" revealed that this atypical anthocyanin accumulation was tightly correlated with abnormal overexpression of the gene-encoding gibberellin (GA) 2-beta-dioxygenase 8, PbGA2ox8. Consistently, decreased methylation was also observed in the promoter region of PbGA2ox8 from "Red Zaosu" compared with "Zaosu". Moreover, the GA levels in "Red Zaosu" seedlings were lower than those in "Zaosu" seedlings, and the application of exogenous GA4 reduced abnormal anthocyanin accumulation in "Red Zaosu". Transient overexpression of PbGA2ox8 reduced the GA4 level and caused anthocyanin accumulation in pear fruit skin. Moreover, the presence of red stripes indicated anthocyanin accumulation in the hypanthial epidermal layer near vascular branches (VBs) in "Red Zaosu". Transient overexpression of PbGA2ox8 resulting from vacuum infiltration induced anthocyanin accumulation preferentially in calcium-enriched areas near the vascular bundles in pear leaves. We propose a fruit-striping mechanism, in which the abnormal overexpression of PbGA2ox8 in "Red Zaosu" induces the formation of a longitudinal array of anthocyanin stripes near vascular bundles in fruit.

Effects of Melatonin Treatment of Postharvest Pear Fruit on Aromatic Volatile Biosynthesis.

Aroma affects the sensory quality of fruit and, consequently, consumer satisfaction. Melatonin (MT) is a plant growth regulator used to delay senescence in postharvest fruit during storage; however, its effect on aroma of pear fruit remains unclear. In this study, we assessed the effects of 0.1 mmol L-1 MT on volatiles and associated gene expression in the fruit of pear cultivars 'Korla' (Pyrus brestschneideri Rehd) and 'Abbé Fetel' (Pyrus communis L.). MT mainly affected the production of C6 aromatic substances in the two varieties. In 'Korla', MT inhibited expression of PbHPL, and reduced hydroperoxide lyase (HPL) activity and content of hexanal and (E)-hex-2-enal. In contrast, MT inhibited activity of lipoxygenase (LOX), reduced expression of PbLOX1 and PbLOX2, promoted PbAAT gene expression, increased alcohol acyltransferase (AAT) activity, and increased propyl acetate, and hexyl acetate content in 'Abbé Fetel' that similarly led to the reduction in content of hexanal and (E)-hex-2-enal. Content of esters in 'Abbé Fetel' pear increased with increasing postharvest storage period. Although mechanisms differed between the two varieties, effects on aroma volatiles mediated by MT were driven by expression of genes encoding LOX, HPL, and AAT enzymes.

Differences among the Anthocyanin Accumulation Patterns and Related Gene Expression Levels in Red Pears.

Differences in coloration exist among red pear cultivars. Here, we selected six red pear cultivars with different genetic backgrounds to elucidate the characteristics of fruit pigmentation. We detected anthocyanin contents and the expression levels of anthocyanin synthesis-related genes in these cultivars at different stages of fruit development. The anthocyanin contents of all six cultivars showed a rise-drop tendency. Principal component and hierarchical cluster analyses were used to distinguish the types of cultivars and the genes crucial to each anthocyanin accumulation pattern. The six cultivars were divided into three groups. Red Zaosu were clustered into one group, Red Sichou and Starkrimson into another group, and Palacer, Red Bartlett, and 5 Hao clustered into a third group. The expression levels of F3H, UFGT2, MYB10, and bHLH3 were similar among the differential coloration patterns of the six cultivars, suggesting a critical and coordinated mechanism for anthocyanin synthesis. Anthocyanin transporters (GST) and light-responsive genes, such as COP1, PIF3.1, and PIF3.2 played limited roles in the regulation of anthocyanin accumulation. This study provides novel insights into the regulation of anthocyanins synthesis and accumulation in red pears.

PbCOP1.1 Contributes to the Negative Regulation of Anthocyanin Biosynthesis in Pear.

The synthesis of anthocyanin in pear (Pyrus bretschneideri) fruit is regulated by light. However, little is known about the molecular mechanisms of pear fruit coloring mediated by upstream light-signaling regulators. Here, the photoresponse factors CONSTITUTIVE PHOTOMORPHOGENIC (COP) 1.1 and 1.2 were cloned from 'Red Zaosu' peel to study their functions in pear fruit coloring. The overexpression vectors pBI121-PbCOP1.1 and pBI121-PbCOP1.2 were constructed to analyze their effects on anthocyanin synthesis in pear fruit. A protein sequence alignment and phylogenetic tree analysis revealed that PbCOP1 proteins are highly homologous with those of other species. An analysis of tissue differential expression showed that the greatest expression levels of PbCOP1s occurred in the leaves. Their expression levels increased in the leaves during development, when the leaves changed from red to green. The overexpression of PbCOP1s in the peel resulted in reduced anthocyanin synthesis at the injection sites. A quantitative PCR analysis of the injection sites showed that PbCOP1.1 significantly inhibited the expression of the anthocyanin synthesis-related genes CHI, DFR, UFGT2, bHLH3, HY5 and GST. Based on the above results, we hypothesize that PbCOP1.1 is an anthocyanin synthetic inhibitory factor of pear coloration.