Transcriptome and metabolomic analysis to reveal the browning spot formation of ‘ Huangguan ’ pear 1

BACKGROUND
Browning spot (BS) disorders seriously affect the appearance quality of 'Huangguan' pear and cause economic losses. Many studies on BS have mainly focused on physiological and biochemical aspects, and the molecular mechanism remains unclear.


RESULTS
In the present study, the structural characteristics of 'Huangguan' pear with BS were observed via scanning electron microscopy (SEM), the water loss and brown spots were evaluated, and transcriptomic and metabolomics analyses were conducted to reveal the molecular mechanism underlying 'Huangguan' pear skin browning disorder. The results showed that the occurrence of BS was accompanied by a decrease in the wax layer and an increase in lignified cells. Genes related to wax biosynthesis were downregulated in BS, resulting in a decrease in the wax layer in BS. Genes related to lignin were upregulated at the transcriptional level, resulting in upregulation of metabolites related to phenylpropanoid biosynthesis. Expression of calcium-related genes were upregulated in BS. Cold-induced genes may represent the key genes that induce the formation of BS. In addition, the results demonstrated that exogenous NaH2PO4·2H2O and ABA treatment could inhibit the incidence of BS during harvest and storage time by increasing wax-related genes and calcium-related genes expression and increasing plant resistance, whereas the transcriptomics results indicated that GA3 may accelerate the incidence and index of BS.


CONCLUSIONS
The results of this study indicate a molecular mechanism that could explain BS formation and elucidate the effects of different treatments on the incidence and molecular regulation of BS.


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'Huangguan' pear (Pyrus bretschneideri × Pyrus pyrifolia) is an early-and medium-maturing 29 cultivar widely planted in northern China that has a high-quality and exquisite appearance after 30 bagging [1]. However, browning spot (BS) disease often occurs at the surface of 'Huangguan' pear 31 fruits after bagging before harvest or during storage [2]. The symptom of BS is a brown spot at first, and 32 then the brown spot spreads irregularly from the disease spot to the surroundings and becomes darker  [17][18][19]). 48 4 Some researchers believe that the thinning of the wax layer and skin cell wall of pears caused by 49 bagging is the main cause of BS [14]. The adaptability of fruit exocarp to severe environmental changes 50 is reduced, and the development of fruit exocarp is delayed after bagging. It has been reported that BS 51 is closely related to calcium deficiency and phenolic dysregulation in pericarp tissue [7,17,20]. To date, 52 research on BS disease has mainly focused on mineral nutrition (such as Ca [1,3,7,9,15,[21][22][23][24][25]

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This study observed the changes at the site of BS and analyzed the molecular mechanism of BS at 62 the transcriptomic and metabolomic levels. The incidence of BS after treatment with exogenous 63 reagents [NaH2PO4·2H2O (P), abscisic acid (ABA), gibberellin A3 (GA3)] during harvest and storage 64 was investigated. The key genes involved in exocarp formation were also analyzed after treatment, 65 which would provide a basis for the molecular mechanism of BS and clarify the influence of different 66 treatments on the molecular regulation of BS.

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Phenotype characteristics of BS disease of 'Huangguan' pear 69 5 Compared with the normal pear skin (Fig. 1A) of 'Huangguan' pear, the BS-infected skin exhibits an 70 irregular chicken claw shape, and the location of the disease is not fixed (Fig. 1B). BS is a 71 physiological disease with slight depression in the affected area. Paraffin section observation revealed 72 that the degree of lignification of the exocarp cells of BS parts was significantly higher than that of the 73 normal parts (Fig. 1 B).C, D). Scanning electron microscopy (SEM) revealed a thick cuticular layer on 74 the skin of the normal 'Huangguan' pear, but the BS part of 'Huangguan' pear skin consisted of layers 75 of dead cells, and exocarp cells were more densely arranged (Fig. 1 B). E, F). Therefore, the occurrence 76 of BS may be caused by necrosis of the exocarp and hypodermal cortical tissues.

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Observation of the paraffin sections of the normal part (C) and BS disease part (D) of 'Huangguan 79 pear'. SEM analysis of the normal part (E) and BS disease part (F) of 'Huangguan' pear.

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Alternatively, we found many cracks on the fruit surface, while the cracks on the BS part 81 disappeared (Additional file 1: Figure. S1). Thus, the experiment to detect RWL between CK and BS 82 was conducted. After 10 d of storage at room temperature (25°C), the RWLs of the three groups of 83 'Huangguan' pear and 'Huangguan' pear with BS disease were calculated. The results showed that the 84 RWLs of two groups (#1 and #3) of 'Huangguan' pear fruits were significantly higher than that of 85 'Huangguan' pear with BS disease (Fig. 2). The average RWLs of the CK and BS groups were 3.49% 86 and 3.19%, respectively. This means that BS lesions could inhibit water loss, which may be regulated 87 by the layers of densely arranged dead cells at the fruit surface.

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The ordinate represents three different groups, and each group has 10 fruits.   Figure S3). KEGG pathway annotation analysis showed that global 113 and overview maps, carbohydrate metabolism, signal transduction and environmental adaptation were 114 overrepresented (Additional file 1: Figure S4). KEGG enrichment analysis was further used to assess 115 DEGs between CK and BS. We found seven significant pathways, including the MAPK signaling    3D). In addition, the up-and downregulation of differential metabolites are 142 listed in Table 2. 143 Cutin, suberine and wax biosynthesis 11 11 0 map00073

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The phenotypic characteristics and metabonomics analysis of the pericarp indicated that the cutin 146 suberin and wax biosynthesis pathway and lignin biosynthesis may be involved in the formation of BS.    Table S3 and Table S4.

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Combined analysis of the metabolome and transcriptome 222 MixOmics[31] multifunctions were used for multivariable dimensionality reduction to explore the 223 relationship between transcriptomics and metabolomics (Fig. 8A). The block.splsda function in 224 mixOmics was used to analyze differential genes and differential metabolites, and plotVar and 225 circosPlot functions were used to visualize the results. We found that there was a closely related

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Blue represents a negative correlation, and red represents a positive correlation.

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Treatments with P, ABA, and GA3 were performed to investigate their effects on the BS of 239 'Huangguan' pear (Fig. 9A, B). P and ABA treatments can significantly reduce the incidence and index 240 of BS. The incidence and index of BS treated with GA3 was higher than those of other treatments. The 241 results showed that P treatment has the best inhibitory effect on BS disorder, and ABA also has a 242 certain inhibitory effect on BS, while GA3 treatment was able to promote the occurrence of BS.

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In addition, we investigated the BS incidence of 'Huangguan' pears with different treatments 244 during storage (Fig. 9C, D, E). We found that P treatment effectively inhibited BS at 4 and 5 months of 245 storage (Fig. 9C, D). ABA treatment inhibited BS at 5 months of storage, while there was no significant 246 difference in other time periods compared with CK (Fig. 9D). The incidence of BS was higher after 247 GA3 treatment during storage, indicating that GA3 may cause the occurrence of BS after 248 20 low-temperature storage (Fig. 9C, D, E). showed that global and overview maps, carbohydrate metabolism, signal transduction and 262 environmental adaptation were overrepresented (Fig. 10C). Furthermore, we identified expression of 263 genes involved in BS formation (Fig 10B)   277 Therefore, we analyzed the expression of five wax-related genes in the pericarp of 'Huangguan' pear 278 after different treatments (Fig. 11) expression of PGIP was increased after ABA treatment. However, GA3 treatment did not affect the 289 expression of these genes and even had a persistent effect (Fig. 11). These results are consistent with 290 transcriptome data. However, a small amount of BS disorder has also been found on unbagged fruits, 317 although the shape of the disease is mostly circular and not consistent with that of bagging (Additional 318 file 1: Figure S5). Therefore, bagging may not be the only cause of BS. We observed that the onset of 319 BS was characterized by a close arrangement of lignified dead cells accompanied by a significant 320 reduction in epidermal wax (Fig. 1). Through transcriptomic analysis, it appears that the expression of 321 wax-related genes in BS was decreased, while the expression of lignin-related genes was increased (Fig.   322   4), which was consistent with the observed phenotypic phenomenon. However, the cause of this 323 phenomenon is still unclear.