Mutations in PmUFGT3 contribute to color variation of fruit skin in Japanese apricot (Prunus mume Sieb. et Zucc.).

BACKGROUND: Japanese apricot (Prunus mume Sieb. et Zucc.) is popular for both ornamental and processing value, fruit color affects the processing quality, and red pigmentation is the most obvious phenotype associated with fruit color variation in Japanese apricot, mutations in structural genes in the anthocyanin pathway can disrupt the red pigmentation, while the formation mechanism of the red color trait in Japanese apricot is still unclear.  RESULTS: One SNP marker (PmuSNP_27) located within PmUFGT3 gene coding region was found highly polymorphic among 44 different fruit skin color cultivars and relative to anthocyanin biosynthesis in Japanese apricot. Meantime, critical mutations were identified in two alleles of PmUFGT3 in the green-skinned type is inactivated by seven nonsense mutations in the coding region, which leads to seven amino acid substitution, resulting in an inactive UFGT enzyme. Overexpression of the PmUFGT3 allele from red-skinned Japanese apricot in green-skinned fruit lines resulted in greater anthocyanin accumulation in fruit skin. Expression of same allele in an Arabidopsis T-DNA mutant deficient in anthocyanidin activity the accumulation of anthocyanins. In addition, using site-directed mutagenesis, we created a single-base substitution mutation (G to T) of PmUFGT3 isolated from green-skinned cultivar, which caused an E to D amino acid substitution and restored the function of the inactive allele of PmUFGT3 from a green-skinned individual. CONCLUSION: This study confirms the function of PmUFGT3, and provides insight into the mechanism underlying fruit color determination in Japanese apricot, and possible approaches towards genetic engineering of fruit color.

miR169 and PmRGL2 synergistically regulate the NF-Y complex to activate dormancy release in Japanese apricot (Prunus mume Sieb. et Zucc.).

KEY MESSAGE: This study is the first to demonstrate that GA4-induced dormancy release is associated with the NF-Y complex, which interacts with gibberellin inhibitor RGL2 in Japanese apricot. Seasonal dormancy is not only vital for the survival in cold winter but also affects flowering of temperate fruit trees and the dormancy release depends on the accumulation of the cold temperatures (Chilling requirement-CR). To understand the mechanism of dormancy release in deciduous fruit crops, we compared miRNA sequencing data during the transition stage from paradormancy to dormancy release in the Japanese apricot and found that the miR169 family showed significant differentially up-regulated expression during dormancy induction and was down-regulated during the dormancy release periods. The 5' RACE assay and RT-qPCR validated its target gene NUCLEAR FACTOR-Y subunit A (NF-YA), which exhibited the opposite expression pattern. Further study showed that exogenous GA4 could inhibit the expression of the gibberellic acid (GA) signal transduction suppressor PmRGL2 (RGA-LIKE 2) and promote the expression of NF-Y. Moreover, the interaction between the NF-Y family and GA inhibitor PmRGL2 was verified by the yeast-two-hybrid (Y2H) system and a bimolecular fluorescence complementarity (BiFC) experiment. These results suggest that synergistic regulation of the NF-Y and PmRGL2 complex leads to the activation of dormancy release induced by GA4. These findings will help to elucidate the functional and regulatory roles of miR169 and NF-Y complex in seasonal bud dormancy induced by GA in Japanese apricot and provide new insights for the discovery of dormancy release mechanisms in woody plants.

Comprehensive transcriptome profiling to identify genes involved in pistil abortion of Japanese apricot.

Flower development exists as a key period in the angiosperms life cycle and the proper development is considered with its reproductive success. Pistil abortion is one of the widely distributed aspects of berry plants and its basic mechanism in Japanese apricot is quite unclear and needs thorough investigation. The present study was carried out to get a deep insight into the pistil abortion mechanism in Japanese apricot using a transcriptomic approach. A large number of DEGs were identified from different development stages of normal and abortive pistils. Pair-wise comparison analysis was performed as LY1 vs DQD1, LY2 vs DQD2, and LY3 vs DQD3 and produced 3590, 2085, and 2286 transcripts, respectively. The Gene Ontology (GO) showed that different metabolic processes, plant hormones, developmental processes, and photosystem-related genes were involved in pistil abortion. The pathway analysis revealed significant enrichment of plant hormone's signal transduction and circadian rhythm pathways. Furthermore, transcription factors such as MYB, MADS-box, and NAC family showed lower expression in abortive pistils. The current study presents a new strategy for advanced research and understanding of the pistil abortion process in Japanese apricot and provides a possible reference for other deciduous fruit trees.

The role of miR319a and its target gene TCP4 in the regulation of pistil development in Prunus mume.

The microRNAs (miRNAs) comprise a broad class of non-coding small endogenous RNAs that are associated with many biological processes through the regulation of target genes, such as leaf morphogenesis and polarity, biotic and abiotic stress responses, and root and flower development. We identified a miRNA that affects flower development, miR319a, in Prunus mume. The Pm-miR319a target, Pm-TCP4, was validated by 5'RACE. The higher expression of Pm-TCP4 in imperfect flowers showed that Pm-TCP4 might promote pistil abortion. Further experiments showed that Pm-miR319a negatively regulates the expression of Pm-TCP4 mRNAs and affected pistil development. Sixteen downstream genes of Pm-TCP4 related to flower development were predicted. Previous studies have shown that they have an impact on the development of pistils. In this study it was established that Pm-miR319a indirectly regulates the development of pistils by regulating its target gene Pm-TCP4.

Genome-wide analysis of PmTCP4 transcription factor binding sites by ChIP-Seq during pistil abortion in Japanese apricot.

The TCP4 transcription factor plays an important role in plant growth and development, especially in flower development. PmTCP4 is involved in the process of pistil abortion in Japanese apricot, but its molecular mechanism, particularly the DNA binding sites and co-regulatory genes, are quite unknown. Therefore, to identify the genome-wide binding sites of PmTCP4 transcription factors and their co-regulatory genes, chromatin immunoprecipitation sequencing (ChIP-Seq) was carried out. ChIP-Seq data produced the maximum enriched peaks in two Japanese apricot cultivars 'Daqiandi' (DQD) and 'Longyan' (LY), which showed that the majority of DNA-protein interactions are relevant and have a significant function in binding sites. Moreover, 720 and 251 peak-associated genes regulated by PmTCP4 were identified in DQD and LY, respectively, and most of them were involved in the flower and pistil development process. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that photosynthesis and oxidative phosphorylation were the most enriched pathways in both cultivars and all identified genes related to these pathways were down-regulated. This study will provide a reference for a better understanding of the PmTCP4 regulatory mechanism during pistil abortion in Japanese apricot.

Identification and expression profiling of sugar transporter genes during sugar accumulation at different stages of fruit development in apricot.

Sugars are considered as an essential signaling molecule for fruit growth and development, which plays a key role in fruit quality. Up to now, the mechanism controlling sugar metabolism and transport in apricot is unclear. Therefore, in the present study, we measured sugar contents at six different stages of fruit development and ripening, and significant variations were observed throughout these stages. The concentration of glucose and fructose first decreased then increased, sucrose concentration first increased then decreased, while the concentration of sorbitol gradually decreased from growth to maturity. Furthermore, thirty sugar transporter genes related to sucrose synthesis and transport were identified and categorized into different subfamilies based on the phylogenetic analysis. The result of cis-regulatory components showed that under different plant hormones, biotic and abiotic stresses, few elements could be regulated. The correlation analysis showed a higher relationship between ParSuSy5, ParSuSy6, ParSuSy7, and ParFK1 genes and sugar contents, indicating that these genes might have a key role in sugar accumulation and fruit quality. In general, these findings will provide a deep understanding of genomic information and expression profiles of sugar transporter genes, which will contribute toward improvement in fruit quality of apricot.

Analyzing Differentially Expressed Genes and Pathways Associated with Pistil Abortion in Japanese Apricot via RNA-Seq.

Reproduction is a critical stage in the flower development process, and its failure causes serious problems affecting fruit quality and yield. Pistil abortion is one of the main factors in unsuccessful reproduction and occurs in many fruit plants. In Japanese apricot, the problem of pistil abortion is very common and affects fruit quality and plant yield; however, its molecular mechanism is not clearly understood. Therefore, in the current study, we used RNA-Seq to identify the differentially expressed genes (DEGs) and pathways actively involved in pistil abortion. A total of 3882 differentially expressed genes were found after cutoff and pairwise comparison analysis. According to KEGG pathway analysis, plant hormone signaling transduction and metabolic pathways were found most significantly enriched in this study. A total of 60 transcription factor families such as MADS-box, NAC and TCP showed their role in this process. RT-qPCR assays confirmed that the expression levels were consistent with RNA-Seq results. This study provides an alternative to be considered for further studies and understanding of pistil abortion processes in Japanese apricot, and it provides a reference related to this issue for other deciduous fruit crops.

Comparative analysis of the complete chloroplast genome among Prunus mume, P. armeniaca, and P. salicina.

Prunus mume Sieb. et Zucc., P. armeniaca L., and P. salicina L. are economically important fruit trees in temperate regions. These species are taxonomically perplexing because of shared interspecific morphological traits and variation, which are mainly attributed to hybridization. The chloroplast is cytoplasmically inherited and often used for evolutionary studies. We sequenced the complete chloroplast genomes of P. mume, P. armeniaca, and P. salicina using Illumina sequencing followed by de novo assembly. The three chloroplast genomes exhibit a typical quadripartite structure with conserved genome arrangement, structure, and moderate divergence. The lengths of the genomes are 157,815, 157,797, and 157,916 bp, respectively. The length of the large single-copy region (LSC) region is 86,113, 86,283, and 86,122 bp, and the length of the SSC region is 18,916, 18,734, and 19,028 bp; the IR region is 26,393, 26,390, and 26,383 bp, respectively. Each of the three chloroplast genomes encodes 133 genes, including 94 protein-coding, 31 tRNA, and eight rRNA genes. Differential gene analysis for the three species revealed that trnY-ATA is a unique gene in P. armeniaca; in contrast, the gene trnI-TAT is only present in P. mume and P. salicina, though the position of the gene in these chloroplast genomes differs. Further comparative analysis of the complete chloroplast genome sequences revealed that the ORF genes and the sequences of linked regions rps16 and atpA, atpH and atpI, trnc-GCA and psbD, ycf3 and atpB, and rpL32 and ndhD are significantly different and may be used as molecular markers in taxonomic studies. Phylogenetic evolution analysis of the three species suggests that P. mume has a closer genetic relationship to P. armeniaca than to P. salicina.

Integrating proteomic and transcriptomic analyses of loquat (Eriobotrya japonica Lindl.) in response to cold stress.

The expression levels of many genes and the related proteins change and regulate physiological and metabolic processes that help the plant survive harsh environmental conditions under cold stress. Damage due to cold and freezing conditions often causes dynamic loss of loquat fruits in cultivated parts of northern China. To illustrate the mechanism of cold tolerance in the loquat, we combined the transcriptomic analysis with isobaric tags for relative and absolute quantification (iTRAQ) and RNA sequencing (RNA-Seq) data from loquat leaves under 4 °C treatment. The results showed 122,081 genes and 1210 differentially expressed genes (DEGs), while only 4582 proteins and 300 differential proteins (DEPs) were identified. Functional annotation and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis indicated that metabolic pathways and biosynthesis of secondary metabolites were the two most common pathways in transcriptional and translational processes in this study. Comparison analysis of the transcriptomic and proteomic profiles, only 27 of 3620 genes were found to be shared both in DEGs and DEPs. Further validation with Real-Time Quantitative RT-PCR analysis showed that the genes expression of NADP-dependent D-sorbitol-6-phosphate dehydrogenase, anthocyanin synthase and phenylalanine ammonia-lyase were consistent with the pattern of transcriptome profile, which suggested that these three genes might play vital roles in cold tolerance in loquat.

Candidate genes associated with red colour formation revealed by comparative genomic variant analysis of red- and green-skinned fruits of Japanese apricot (Prunus mume).

The red-skinned fruit of Japanese apricot (Prunus mume Sieb. et Zucc) appeals to customers due to its eye-catching pigmentation, while the mechanism related to its colour formation is still unclear. In this study, genome re-sequencing of six Japanese apricot cultivars was carried out with approximately 92.2 Gb of clean bases using next-generation sequencing. A total of 32,004 unigenes were assembled with an average of 83.1% coverage rate relative to reference genome. A wide range of genetic variation was detected, including 7,387,057 single nucleotide polymorphisms, 456,222 insertions or deletions and 129,061 structural variations in all genomes. Comparative sequencing data revealed that 13 candidate genes were involved in biosynthesis of anthocyanin. Significantly higher expression patterns were observed in genes encoding three anthocyanin synthesis structural genes (4CL, F3H and UFGT), five transcription factors (MYB-bHLH-WD40 complexes and NAC) and five anthocyanin accumulation related genes (GST1, RT1, UGT85A2, ABC and MATE transporters) in red-skinned than in green-skinned Japanese apricots using reverse transcription-quantitative polymerase chain reaction. Eight main kinds of anthocyanin s were detected by UPLC/MS, and cyanidin 3-glucoside was identified as the major anthocyanin (124.2 mg/kg) in red-skinned cultivars. The activity of UDP-glucose flavonoid-3-O-glycosyltransferase enzyme determined by UPLC was significantly higher in all red-skinned cultivars, suggesting that it is the potential vital regulatory gene for biosynthesis of anthocyanin in Japanese apricot.

UFGT: The Key Enzyme Associated with the Petals Variegation in Japanese Apricot.

Japanese apricot (Prunus mume Sieb.et Zucc.) is an important ornamental plant in China. One of the traits of petals color variegation is attractive, but its formation mechanism is unclear. In our study, RNA-seq technology was employed to characterize the transcriptome response to the mutation of "Fuban Tiaozhi" associated with petals variegation in Japanese apricot. As a result, 4,579,040 (white-flowered, WF) and 7,269,883 (red-flowered, RF) reads were mapped to P. persica genes, while 5,006,676 (WF) and 7,907,436 (RF) were mapped to P. persica genomes. There were 960 differentially expressed genes (DEGs) identified. Gene ontology analysis showed that these genes involved in 37 functional groups including 19 biological processes, 10 cellular components and eight molecular functions. Pathway enrichment annotation demonstrated that highly ranked genes were associated with flavonoid biosynthesis, anthocyanin biosynthesis, anthocyanins transports, plant hormone signal transduction, and transcriptional factors. The expression patterns part of them were validated by qRT-PCR. We found that UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) gene showed differential expression pattern. The UFGT enzyme activities in RF had a significantly higher than that of WF and lower in the initial stage and increased when the red appeared in the petals, which is identical to the accumulation of anthocyanins. And we also validated the SNPs, leading to the nonsynonymous mutations, in the UFGT by Sanger sequencing which may affect the enzyme activity. In summary, our results provide molecular candidates for better understanding the mechanisms of the variegation in Japanese Apricot.

Driver fatigue detection based on eye state.

BACKGROUND: Nowadays, more and more traffic accidents occur because of driver fatigue. OBJECTIVE: In order to reduce and prevent it, in this study, a calculation method using PERCLOS (percentage of eye closure time) parameter characteristics based on machine vision was developed. It determined whether a driver's eyes were in a fatigue state according to the PERCLOS value. METHODS: The overall workflow solutions included face detection and tracking, detection and location of the human eye, human eye tracking, eye state recognition, and driver fatigue testing. The key aspects of the detection system incorporated the detection and location of human eyes and driver fatigue testing. The simplified method of measuring the PERCLOS value of the driver was to calculate the ratio of the eyes being open and closed with the total number of frames for a given period. RESULTS: If the eyes were closed more than the set threshold in the total number of frames, the system would alert the driver. CONCLUSION: Through many experiments, it was shown that besides the simple detection algorithm, the rapid computing speed, and the high detection and recognition accuracies of the system, the system was demonstrated to be in accord with the real-time requirements of a driver fatigue detection system.