Integrated analysis of HSP20 genes in the developing flesh of peach: identification, expression profiling, and subcellular localization.

BACKGROUND: Plant HSP20s are not only synthesized in response to heat stress but are also involved in plant biotic and abiotic stress resistance, normal metabolism, development, differentiation, survival, ripening, and death. Thus, HSP20 family genes play very important and diverse roles in plants. To our knowledge, HSP20 family genes in peach have not yet been characterized in detail, and little is known about their possible function in the development of red flesh in peach. RESULTS: In total, 44 PpHSP20 members were identified in the peach genome in this study. Forty-four PpHSP20s were classified into 10 subfamilies, CI, CII, CIII, CV, CVI, CVII, MII, CP, ER, and Po, containing 18, 2, 2, 10, 5, 1, 1, 2, 1, and 2 proteins, respectively. Among the 44 PpHSP20 genes, 6, 4, 4, 3, 7, 11, 5, and 4 PpHSP20 genes were located on chromosomes 1 to 8, respectively. In particular, approximately 15 PpHSP20 genes were located at both termini or one terminus of each chromosome. A total of 15 tandem PpHSP20 genes were found in the peach genome, which belonged to five tandemly duplicated groups. Overall, among the three cultivars, the number of PpHSP20 genes with higher expression levels in red flesh was greater than that in yellow or white flesh. The expression profiling for most of the PpHSP20 genes in the red-fleshed 'BJ' was higher overall at the S3 stage than at the S2, S4-1, and S4-2 stages, with the S3 stage being a very important period of transformation from a white color to the gradual anthocyanin accumulation in the flesh of this cultivar. The subcellular localizations of 16 out of 19 selected PpHSP20 proteins were in accordance with the corresponding subfamily classification and naming. Additionally, to our knowledge, Prupe.3G034800.1 is the first HSP20 found in plants that has the dual targets of both the endoplasmic reticulum and nucleus. CONCLUSIONS: This study provides a comprehensive understanding of PpHSP20s, lays a foundation for future analyses of the unknown function of PpHSP20 family genes in red-fleshed peach fruit and advances our understanding of plant HSP20 genes.

Differences in PpAAT1 Activity in High- and Low-Aroma Peach Varieties Affect γ-Decalactone Production.

Aroma contributes to the unique flavors of fruits and is important for fruit quality evaluation. Among the many volatiles in peach (Prunus persica) fruits, γ-decalactone has the greatest contribution to the characteristic peach aroma. Some peach cultivars have γ-decalactone contents that are too low to detect. Comparison of the transcriptomes and metabolomes of a high-aroma cultivar, Fenghuayulu, and a low-aroma cultivar, Achutao, suggested that amino acid substitutions in ALCOHOL ACYLTRANSFERASE (PpAAT1) are responsible for the undetectable levels of γ-decalactone in cv Achutao fruit. Modeling and molecular docking analysis of PpAAT1 indicated that the substituted residues might determine substrate recognition or act as control channels to the active site. In vitro enzyme assays on PpAAT1 heterologously expressed and purified from Escherichia coli and in vivo assays using transient PpAAT1 expression in Nicotiana benthamiana or the oleaginous yeast Yarrowia lipolytica indicated that PpAAT1 from high-aroma cultivars was more efficient than PpAAT1 from low-aroma cultivars in catalyzing the conversion of 4-hydroxydecanoyl-coenzyme A into γ-decalactone. Examination of loss-of-function mutations of PpAAT1 generated by CRISPR/Cas9 in cv Fenghuayulu showed that fruits with PpAAT1 mutations had significantly lower γ-decalactone contents. Expression of the version of PpAAT1 from cv Fenghuayulu in cv Achutao restored γ-decalactone levels to those measured in 'Fenghuayulu', confirming the specific contribution of PpAAT1 to the formation of this key aroma compound. These results show how the biosynthesis of the peach aroma compound γ-decalactone is compromised in some low-aroma cultivars and illustrate the physiological role of PpAAT1 in plant lactone biosynthesis.

Transcriptomic Analysis Reveals Genes Associated with the Regulation of Peach Fruit Softening and Senescence during Storage.

Peach (Prunus persica (L.) Batsch) is a highly desirable fruit that is consumed around the world. However, the peach fruit is highly perishable after harvest, a characteristic that limits the distribution and supply to the market and causes heavy economic losses. Thus, peach fruit softening and senescence after harvest urgently need to be addressed. In the current study, transcriptomic analysis was performed to identify candidate genes associated with peach fruit softening and senescence, comparing peach fruit from cultivars with different flesh textures, namely melting and stony hard (SH) flesh textures during storage at room temperature. The mitogen-activated protein kinase signaling pathway-plant and plant hormone signal transduction pathways were associated with peach fruit softening and senescence according to the Venn diagram analysis and weighted gene co-expression network analysis. The expression levels of seven genes, including Prupe.1G034300, Prupe.2G176900, Prupe.3G024700, Prupe.3G098100, Prupe.6G226100, Prupe.7G234800, and Prupe.7G247500, were higher in melting peach fruit than in SH peach fruit during storage. Furthermore, the SH peach fruit softened rapidly after 1-naphthylacetic acid treatment, during which the levels of expression of these seven genes, determined by a quantitative reverse transcription polymerase chain reaction, were strongly induced and upregulated. Thus, these seven genes may play essential roles in regulating peach fruit softening and senescence.

Genome-wide identification and classification of MYB superfamily genes in peach.

The MYB transcription factor superfamily is one of the largest superfamilies modulating various biological processes in plants. Over the past few decades, many MYB superfamily genes have been identified and characterized in some plant species. However, genes belonging to the MYB superfamily in peach (Prunus persica) have not been comprehensively identified and characterized although the genome sequences of peach were released several years ago. In total, this study yielded a set of 256 MYB superfamily genes that was divided into five subfamilies: the R2R3-MYB (2R-MYB), R1R2R3-MYB (3R-MYB), MYB-related (1R-MYB), 4R-MYB, and Atypical-MYB subfamilies. These subfamilies contained 128, 4, 109, 1, and 14 members, respectively. The 128 R2R3-MYB subfamily genes in peach were further clustered into 35 groups, and the 109 MYB-related subfamily genes were further clustered into 6 groups: the CCA1-like, CPC-like, TBP-like, I-box-binding-like, R-R-type, and Peach-specific groups. The motif compositions and exon/intron structures within each group within the R2R3-MYB or MYB-related subfamily in peach were highly conserved. The logo sequences of the R2 and R3 repeats of R2R3-MYB subfamily members were highly conserved with those in these repeats of several other plant species. Except for 48 novel peach-specific MYB genes, the remaining 208 out of 256 MYB genes in peach were conserved with the corresponding 198 MYB genes in A. thaliana. Additionally, the 256 MYB genes unevenly distributed on chromosomes 1 to 8 of the peach genome. Eighty-one orthologous pairs of peach/A. thaliana MYB genes were identified among 256 MYB genes in peach and 198 MYB genes in A. thaliana in this study. In addition, 146 pairs of paralogous MYB genes were identified on the eight chromosomes of peach. The expression levels of some of the 51 MYB genes selected for qRT-PCR analysis decreased or increased with red-fleshed fruit development, while the expression patterns of some genes followed no clear rules over the five developmental stages of fruits. This study laid the foundation for further functional analysis of MYB superfamily genes in peach and enriched the knowledge of MYB superfamily genes in plant species.

[Study on preparation process and analytical methods of ESAC from Ganoderma lucidum].

OBJECTIVE: To develop the procedure for separating the ethanol-soluble and acidic components (ESAC) from Ganoderma lucidum, and to establish a method for quantifying ESAC in G. lucidum. METHOD: The ethanol extract of G. lucidum was extracted with a saturated NaHCO3 solution, acidified and re-extracted by chloroform to obtain ESAC. The quantitative analysis of ESAC was based on the characteristic color reaction between ESAC and H2SO4. RESULT: The optimal conditions for separating ESAC on a 10 g scale are as follows: ratio of material and ethanol (mL), 1:15; immersing time, 24 h; volume of saturated NaHCO3 and chloroform, 1300 mL; extract 3 times. The condition for measuring ESAC is as follows: sample weight, 1 g; solution volume, 1.5 mL; immuersing time, 0.5 h; detecting reagent, 50% H2SO4 in ethanol; heating time in 100 degrees C water bathe, 3 min; measuring wavelength, 490 nm. CONCLUSION: The procedure for ESAC preparation is simple and well-designed, and the established method for ESAC can be used for the qualitative analysis of the G. lucidum related products.