Calcium acts as a secondary messenger in plants and is essential for plant growth and development. However, studies on the pathway of aroma synthesis in \'Nanguo\' pear (Pyrus ussriensis Maxim.) are scarce. In this study, a bioinformatics analysis of transcriptomic data from calcium-treated \'Nanguo\' pear was performed, which identified two fatty acid desaturases, PuFAD2 and PuFAD3, and eight AP2/ERF transcription factors, all exhibiting the same expression patterns. Transient expression experiments showed overexpression of PuFAD2 and PuFAD3 significantly increased the levels of aromatic substrates linoleic acid, hexanal, linolenic acid, and (E)-2-hexenal, but RNAi (RNA interference) had the opposite expression. Promoter sequences analysis revealed that PuFAD2 and PuFAD3 have ERE (estrogen response element) motifs on their promoters. The strongest activation of PuFAD2 by PuERF008 was verified using a dual-luciferase reporting system. Additionally, yeast one-hybrid and electrophoretic mobility shift assays revealed PuERF008 could active PuFAD2. Transient overexpression and RNAi analyses of PuERF008 showed a strong correlation with the expression of PuFAD2. This study provides insights into the process of aroma biosynthesis in \'Nanguo\' pear and offers a theoretical basis for elucidating the role of calcium signaling in aroma synthesis.

Volatile esters are the main aromatic components that affect consumer sensory preferences. Aroma is a crucial characteristic of the \'Nanguo\' pear (Pyrus ussriensis Maxim). Carboxylesterases (CXEs) are positively correlated with the catabolism of volatile esters in peaches; however, the mechanism of action of CXE family members in \'Nanguo\' pear is poorly understood. In this study, 40 PuCXEs were identified in the \'Nanguo\' pear and assigned into seven groups. In addition, we found that most PuCXEs were relatively conserved and contained cytoplasmic proteins. This hypothesis was supported by phylogenetic analysis, investigation of conserved domains and gene structures, and prediction of subcellular localization. Based on the content of volatile esters and expression levels of PuCXEs analysis, four PuCXEs, including PuCXE7, PuCXE15, PuCXE20, and PuCXE25, had a significant negative correlation with volatile ester accumulation. Particularly, the correlation of PuCXE15 far exceeded that of the other PuCXEs. The results of the transient expression assay showed that PuCXE15 promoted the degradation of ester in vivo. Subcellular localization experiment revealed that PuCXE15 is located in the plasma membrane and nucleus. These results show that PuCXE15 functions in the catabolism of volatile ester in \'Nanguo\' pear fruit, and provides a foundation for enhancing aroma quality by artificial control in pear.

Long-term storage of pear fruit at low temperature can retard senescence but may result in pericarp browning. We previously reported that increasing endogenous γ-aminobutyrate (GABA) content by exogenous GABA treatment can maintain mitochondrial structure integrity, thereby alleviating pericarp browning of \'Nanguo\' pears after cold storage. Here, we tested the effectiveness of Ca2+ treatment on pericarp browning in relation to GABA biosynthesis. Fruit browning was reduced by treatment with Ca2+ after 180 days of storage. Pericarp Ca2+ and calmodulin content in treated fruit increased, and concomitantly, endogenous GABA content, key GABA synthesis-related enzyme activity, and gene expression were upregulated. Moreover, the mitochondrial structure in the pericarp tissue was found to be well preserved. Thus, Ca2+ treatment effectively reduced pericarp browning of refrigerated \'Nanguo\' pears owing to improvement in the GABA biosynthesis capacity in the fruit.

Cold storage is widely used for delaying ripening and senescence; however, fruit aroma diminishes noticeably after long-term cold storage. The esters synthesized by the lipoxygenase (LOX) pathway are responsible for \'Nanguo\' pear aroma. As methyl jasmonate (MeJA) is known to act on various fruit qualities, we investigated whether it acts via the LOX pathway in cold-stored \'Nanguo\' pears. MeJA treatment increased the content of volatile esters and unsaturated fatty acids and the activities of alcohol acyltransferase, alcohol dehydrogenase, and LOX. It also up-regulated the expression of key genes (PuAAT, PuADH3, PuADH5, PuADH9, PuLOX1, and PuLOX3) in the LOX pathway and that of transcription factors (PuMYB21-like, PuMYB108-like, PuWRKY61, PuWRKY72, and PuWRKY31), whose genes were differentially expressed in preliminary transcriptome analysis. Therefore, considering its effects on LOX pathway-related genes and transcription factors, MeJA may be useful in preventing cold-storage-induced decline in ester biosynthesis, aroma, and consequently the quality of cold-stored \'Nanguo\' pears.

\'Nanguo\' pear is particularly renowned for its fragrance. Esters are the main components of its aroma, which are synthesized primarily by the LOX pathway. We identified the main volatile esters and critical gene family members involved in the LOX pathway by monitoring their variation accompanying post-harvest ripening and examining their roles through principal component analysis (PCA), partial least-square regression (PLSR), and correlation analysis. In pears ripening to the optimum taste period (OTP), components and contents of volatile esters reached a peak, of which ethyl butanoate, ethyl hexanoate, and hexyl acetate were most prominent. Linoleic acid and linolenic acid contents rose greatly until OTP and then declined; the activities of LOX, alcohol dehydrogenase (ADH), and alcohol acyltransferase (AAT) increased progressively until the OTP. Among the genes involved in LOX-pathway, the expressions of PuLOX3, PuADH3, and PuAAT contributed most to changes of total ester and main esters in \'Nanguo\' pears.

Although refrigeration is commonly used in the storage of \'Nanguo\' pears (Pyrus ussuriensis Maxim.), long-term refrigeration can result in browning of the pericarp. In this study, we aimed to determine how γ-aminobutyrate (GABA) affects the mitochondrial oxidation defense system in \'Nanguo\' pears and how it might be used to prevent post-refrigeration peel browning. We found that fruit treated with GABA browned slower, and had lower browning indices and reactive oxygen and malondialdehyde content; increased peroxidase, superoxide dismutase, alternating oxidase, and catalase enzyme activities; and heightened enzyme-related gene expression. The mitochondria of GABA-treated fruit also showed less damage following cold storage, and there were decreases in mitochondrial membrane permeability transition pore concentrations. Furthermore, we detected an increase in the endogenous GABA content of fruits following GABA treatment. These observations indicate that, by regulating the mitochondrial oxidative defense system and maintaining mitochondrial structure, GABA is effective in terms of reducing peel browning.

Low temperatures retard postharvest ripening and prolong the supply period of \'Nanguo\' pears. However, quality deterioration, or more specifically, a faded aroma occurs in long-term cold-stored fruits. To understand the implicit mechanism, we analyzed the change in content of the main aroma esters and fatty acids by chromatography-mass spectrometry (GC-MS), and the expression patterns of the main genes in fatty acid metabolism pathways. The results showed that hexyl hexanoate disappeared completely and the content of five other aromas declined significantly in cold-stored fruit during the optimal taste period. The content of saturated and unsaturated fatty acids significantly increased and decreased, respectively in cold-stored fruit. fadD, fadE, fadJ, atoB, fabF, SCD, FAD2, LOX2S, LOX1_5 and HPL genes down-regulated in fatty acid metabolism during shelf life in the fruit after cold storage. In conclusion, the faded aroma of cold-stored fruit was mainly regulated by fatty acid metabolism pathway.

Cold storage is commonly employed to delay senescence in \'Nanguo\' pear after harvest. However, this technique also causes fruit aroma weakening. MicroRNAs are regulators of gene expression at the post-transcriptional level that play important roles in plant development and in eliciting responses to abiotic environmental stressors. In this study, the miRNA transcript profiles of the fruit on the first day (C0, LT0) move in and out of cold storage and the optimum tasting period (COTP, LTOTP) during shelf life at room temperature and after cold storage were analyzed, respectively. 314 known miRNAs were identified in \'Nanguo\' pear; 176 and 135 miRNAs were significantly differentially expressed on the C0 vs. LT0 and on the COTP vs. LTOTP, respectively. After prediction the target genes of these differentially expressed miRNAs, 9 s-lipoxygenase (LOX2S), 13 s-lipoxygenase (LOX1_5), hydroperoxide lyase (HPL), and alcohol dehydrogenase (ADH1) were found differentially expressed, which were the key genes during aroma formation. The expression pattern of these target genes and the related miRNAs were identified by RT-PCR. mdm-miR172a-h, mdm-miR159a/b, mdm-miR160a-e, mdm-miR395a-i, mdm-miR399a/b/c, mdm/ppe-miR535a/b, and mdm-miR7120a/b may negatively regulate the target genes expression. These results indicate that miRNAs may play key roles in aroma weakening in cold storage \'Nanguo\' pear and provide valuable information for studying the molecular mechanisms of miRNAs in the aroma weakening of fruit due to low temperature.

BACKGROUND: Postharvest ripening of the \'Nanguo\' pear (Pyrus ussuriensis Maxim.) can be impeded by low-temperature storage. However, pears after long-term refrigeration are prone to peel browning when returned to room temperature conditions. This study investigated the browning mechanism of \'Nanguo\' pear stored at a low temperature by analysing the differentially expressed proteins between healthy fruit and fruit with peel browning.
RESULTS: The results showed that 181 proteins underwent statistically significant changes. A categorisation of the disparately accumulated proteins was performed using gene ontology annotation. The results showed that the \'metabolic process\', \'cellular process\', \'catalytic activity\', and \'binding\' proteins were the most affected after low-temperature storage. Further analysis revealed that the differentially expressed proteins, which are related to peel browning, are primarily involved in the phenylpropanoid pathway, linoleic acid pathways, fatty acid biosynthesis pathway, glutathione metabolism pathway, photosynthesis pathway, oxidative phosphorylation pathway, and glycolysis pathway.
CONCLUSIONS: This study reveals that there are variations in key proteins in \'Nanguo\' pear after low-temperature storage, and the identification of these proteins will be valuable in future functional genomics studies, as well as provide protein resources that can be used in the efforts to improve pear quality. © 2016 Society of Chemical Industry.