Characterization and Potential Action Mode Divergences of Homologous ACO1 Genes during the Organ Development and Ripening Process between Non-Climacteric Grape and Climacteric Peach.

Ethylene is one crucial phytohormone modulating plants' organ development and ripening process, especially in fruits, but its action modes and discrepancies in non-climacteric grape and climacteric peach in these processes remain elusive. This work is focused on the action mode divergences of ethylene during the modulation of the organ development and ripening process in climacteric/non-climacteric plants. We characterized the key enzyme genes in the ethylene synthesis pathway, VvACO1 and PpACO1, and uncovered that their sequence structures are highly conserved, although their promoters exhibit important divergences in the numbers and types of the cis-elements responsive to hormones, implying various responses to hormone signals. Subsequently, we found the two have similar expression modes in vegetative organ development but inverse patterns in reproductive ones, especially in fruits. Then, VvACO1 and PpACO1 were further validated in promoting fruit ripening functions through their transient over-expression/RNAi-expression in tomatoes, of which the former possesses a weaker role than the latter in the fruit ripening process. Our findings illuminated the divergence in the action patterns and function traits of the key VvACO1/PpACO1 genes in the tissue development of climacteric/non-climacteric plants, and they have implications for further gaining insight into the interaction mechanism of ethylene signaling during the modulation of the organ development and ripening process in climacteric/non-climacteric plants.

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.

Exogenous auxin regulates the growth and development of peach fruit at the expansion stage by mediating multiple-hormone signaling.

BACKGROUND: Fruit expansion stage is crucial to fruit yield and quality formation, and auxin plays a significant role by mediating multi-hormone signals during fruit expansion. However, till now, it is still unclear of the molecular regulatory network during auxin-mediated peach fruit expansion. RESULTS: Here, exogenous NAA application markedly increased IAA content and drastically decreased ABA content at the fruit expansion stage. Correspondingly, NAA mainly induced the auxin biosynthesis gene (1 PpYUCCA) and early auxin-responsive genes (7PpIAA, 3 PpGH3, and 14 PpSAUR); while NAA down-regulated ABA biosynthesis genes (2 PpNCED, 1 PpABA3, and 1 PpAAO3). In addition, many DEGs involved in other plant hormone biosynthesis and signal transduction were significantly enriched after NAA treatment, including 7 JA, 7 CTK, 6 ETH, and 3 GA. Furthermore, we also found that NAA treatment down-regulated most of genes involved in the growth and development of peach fruit, including the cell wall metabolism-related genes (PpEG), sucrose metabolism-related genes (PpSPS), phenylalanine metabolism-related genes (PpPAL, Pp4CL, and PpHCT), and transcription factors (PpNAC, PpMADS-box, PpDof, PpSBP, and PpHB). CONCLUSION: Overall, NAA treatment at the fruit expansion stage could inhibit some metabolism processes involved in the related genes in the growth and development of peach fruit by regulating multiple-hormone signaling networks. These results help reveal the short-term regulatory mechanism of auxin at the fruit expansion stage and provide new insights into the multi-hormone cascade regulatory network of fruit growth and development.

Integrative analysis of the metabolome and transcriptome reveals the molecular mechanism of chlorogenic acid synthesis in peach fruit.

As the most abundant phenolic acid in peach fruit, chlorogenic acid (CGA) is an important entry point for the development of natural dietary supplements and functional foods. However, the metabolic and regulation mechanisms underlying its accumulation in peach fruits remain unclear. In this study, we evaluated the composition and content of CGAs in mature fruits of 205 peach cultivars. In peach fruits, three forms of CGA (52.57%), neochlorogenic acid (NCGA, 47.13%), and cryptochlorogenic acid (CCGA, 0.30%) were identified. During the growth and development of peach fruits, the content of CGAs generally showed a trend of rising first and then decreasing. Notably, the contents of quinic acid, shikimic acid, p-coumaroyl quinic acid, and caffeoyl shikimic acid all showed similar dynamic patterns to that of CGA, which might provide the precursor material basis for the accumulation of CGA in the later stage. Moreover, CGA, lignin, and anthocyanins might have a certain correlation and these compounds work together to maintain a dynamic balance. By the comparative transcriptome analysis, 8 structural genes (Pp4CL, PpCYP98A, and PpHCT) and 15 regulatory genes (PpMYB, PpWRKY, PpERF, PpbHLH, and PpWD40) were initially screened as candidate genes of CGA biosynthesis. Our findings preliminarily analyzed the metabolic and molecular regulation mechanisms of CGA biosynthesis in peach fruit, which provided a theoretical basis for developing high-CGA content peaches in future breeding programs.

Carotenoid Profiling of Yellow-Flesh Peach Fruit.

In this study, the carotenoid profiles and content in 132 cultivars of yellow-flesh peach having different fruit developmental periods (short, middle, and long), fruit surface indumenta (glabrous and pubescent skin), and flesh colors (yellow, golden, and orange) were investigated. We simultaneously analyzed and compared the levels of five carotenoids (lutein, zeaxanthin, ß-cryptoxanthin, α-carotene, and ß-carotene) through high-performance liquid chromatography. Large differences in carotenoid content among germplasms were observed, with coefficients of variation ranging from 21.24% to 67.78%. The carotenoid content, from high to low, was as follows: ß-carotene > zeaxanthin > α-carotene > ß-cryptoxanthin > lutein. We screened several varieties with high carotenoid content, including zeaxanthin in 'Ruiguang2', ß-cryptoxanthin in 'NJN76' and 'TX4F244C', and ß-carotene and total carotenoids in 'Jintong7', '77-26-7', and '77-20-5'. A longer fruit developmental period was associated with greater ß-carotene accumulation but lowered the zeaxanthin and ß-cryptoxanthin accumulation. The zeaxanthin, ß-carotene, and total carotenoid concentrations significantly increased as the flesh color deepened, but the lutein and α-carotene levels remained similar among the three flesh colors. The classification index of the indumenta significantly affected the ß-carotene and total carotenoid content (p < 0.05) and was higher in pubescent than glabrous skin.

DNA and Histone Methylation Regulates Different Types of Fruit Ripening by Transcriptome and Proteome Analyses.

Methylation affects different aspects of genetic material stability, gene expression regulation, and histone modification. The previous reports depicted that DNA and histone methylation regulates plant growth and development. In this study, we evaluated the effects of DNA and histone methylation on 'Hongjia' strawberry and 'Lichun' tomato. We investigated the transient transformation system for arginine methyltransferase (FvPRMT1.5) overexpression and interference and assessed the phenotypic appearance and mRNA and protein expression levels. Results depicted that changes in methylation levels caused inhibition of carotenoids and anthocyanins. Furthermore, the profiling of aroma components was altered in response to 5-azacytidine. DNA hypomethylation induced the expression levels of genes involved in photosynthesis, flavonoid biosynthesis, and hormone signal transduction pathways, while the expression levels of related proteins showed a downward trend. Overall, we proposed a model that reveals the possible regulatory effects of DNA and histone methylation during fruit ripening.

Predictive role of C-reactive protein in sudden death: a meta-analysis of prospective studies.

OBJECTIVE: C-reactive protein (CRP) is a powerful predictor of and risk factor for cardiovascular disease. However, the relationship between CRP and sudden death (SD) is controversial. Therefore, we performed a meta-analysis to evaluate the association between CRP and SD. METHODS: We conducted a comprehensive search of the databases of PubMed, Web of Science, Embase, Cochrane Library, Wanfang, CNKI, China Biology Medicine disc, and Weipu. Two researchers independently screened the literature, extracted data, and evaluated the data quality. The overall effect size was meta-analyzed using Stata software version 12.0 (StataCorp, College Station, TX, USA). RESULTS: Twelve prospective studies involving 36,646 patients were included in the present meta-analysis. The data revealed that patients with higher CRP concentrations had a greater risk of SD (hazard ratio, 1.19; 95% confidence interval, 1.09-1.29). When the hazard ratio of SD was calculated by multivariate analysis of nine studies, CRP was confirmed to be an independent predictive factor for SD (hazard ratio, 1.05; 95% confidence interval, 1.03-1.07). CONCLUSIONS: This meta-analysis confirmed that CRP is an independent predictor of SD. These results support the recommendation of recording the CRP concentration for risk assessment of SD in clinical practice.

Characterization and regulatory mechanism analysis of VvmiR156a-VvAGL80 pair during grapevine flowering and parthenocarpy process induced by gibberellin.

MicroRNA156 (miR156) is an important conserved miRNA family in plants. Recently, we revealed VvmiR156a could involve in the modulation of gibberellin (GA)-mediated flower and berry development process of grapevine (Vitis vinifera L.). However, how to manipulate this process is unclear. For this, we used the GA-induced grapevine parthenocarpy system to investigate the regulatory roles of VvmiR156a during this process. Here, we cloned the mature and precursor sequences of VvmiR156a in Wink grape and identified its potential target gene VvAGL80, which belongs to the MADS-box gene family. Moreover, using RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-RACE) and poly(A)polymerase-mediated 3' rapid amplification of cDNA (PPM-RACE) technologies, it confirmed that VvAGL80 was the true target gene of VvmiR159a. Analysis of promoter cis-elements and ß-glucuronidase (GUS) staining showed that both VvmiR156a and VvAGL80 contained GA-responsive elements and could respond to GA treatments. Quantitative real-time-polymerase chain reaction (qRT-PCR) analysis exhibited the VvmiR156a and VvAGL80 showed opposite expression trends during grapevine flower and berry development, indicating that VvmiR156a negatively regulated the expression of VvAGL80 during this process. After GA treatment, the expression of miR156 in flowers was downregulated significantly, while that of VvAGL80 was upregulated, thereby accelerating grapevine flowering. Furthermore, GA treatment enhanced the negative regulation of VvmiR156a on VvAGL80 in seed, especially at the seed-coat hardening stage, which was the key period of seed growth and development. Our findings enriched the knowledge of the regulatory mechanism of the miRNA-mediated grapevine parthenocarpy process.

Identification of miRNAs-mediated seed and stone-hardening regulatory networks and their signal pathway of GA-induced seedless berries in grapevine (V. vinifera L.).

BACKGROUND: Stone-hardening stage is crucial to the development of grape seed and berry quality. A significant body of evidence supports the important roles of MicroRNAs in grape-berry development, but their specific molecular functions during grape stone-hardening stage remain unclear. RESULTS: Here, a total of 161 conserved and 85 species-specific miRNAs/miRNAs* (precursor) were identified in grape berries at stone-hardening stage using Solexa sequencing. Amongst them, 30 VvmiRNAs were stone-hardening stage-specific, whereas 52 exhibited differential expression profiles during berry development, potentially participating in the modulation of berry development as verified by their expression patterns. GO and KEGG pathway analysis showed that 13 VvmiRNAs might be involved in the regulation of embryo development, another 11 in lignin and cellulose biosynthesis, and also 28 in the modulation of hormone signaling, sugar, and proline metabolism. Furthermore, the target genes for 4 novel VvmiRNAs related to berry development were validated using RNA Ligase-Mediated (RLM)-RACE and Poly(A) Polymerase-Mediated (PPM)-RACE methods, and their cleavage mainly occurred at the 9th-11th sites from the 5' ends of miRNAs at their binding regions. In view of the regulatory roles of GA in seed embryo development and stone-hardening in grape, we investigated the expression modes of VvmiRNAs and their target genes during GA-induced grape seedless-berry development, and we validated that GA induced the expression of VvmiR31-3p and VvmiR8-5p to negatively regulate the expression levels of CAFFEOYL COENZYME A-3-O-METHYLTRANSFERASE (VvCCoAOMT), and DDB1-CUL4 ASSOCIATED FACTOR1 (VvDCAF1). The series of changes might repress grape stone hardening and embryo development, which might be a potential key molecular mechanism in GA-induced grape seedless-berry development. Finally, a schematic model of miRNA-mediated grape seed and stone-hardening development was proposed. CONCLUSION: This work identified 30 stone-hardening stage-specific VvmiRNAs and 52 significant differential expression ones, and preliminary interpreted the potential molecular mechanism of GA-induced grape parthenocarpy. GA negatively manipulate the expression of VvCCoAOMT and VvDCAF1 by up-regulation the expression of VvmiR31-3p and VvmiR8-5p, thereby repressing seed stone and embryo development to produce grape seedless berries.

Characterization and Action Mechanism Analysis of VvmiR156b/c/d-VvSPL9 Module Responding to Multiple-Hormone Signals in the Modulation of Grape Berry Color Formation.

In recent years, more and more reports have shown that the miR156-SPL module can participate in the regulation of anthocyanin synthesis in plants. However, little is known about how this module responds to hormonal signals manipulating this process in grapes. In this study, exogenous GA, ABA, MeJA, and NAA were used to treat the 'Wink' grape berries before color conversion, anthocyanin and other related quality physiological indexes (such as sugar, aroma) were determined, and spatio-temporal expression patterns of related genes were analyzed. The results showed that the expression levels of VvmiR156b/c/d showed a gradually rising trend with the ripening and color formation of grape berries, and the highest expression levels were detected at day 28 after treatment, while the expression level of VvSPL9 exhibited an opposite trend as a whole, which further verifies that VvmiR156b/c/d can negatively regulate VvSPL9. Besides, VvmiR156b/c/d was positively correlated with anthocyanin content and related genes levels, while the expression pattern of VvSPL9 showed a negative correlation. Analysis of promoter cis-elements and GUS staining showed that VvmiR156b/c/d contained a large number of hormone response cis-elements (ABA, GA, SA, MeJA, and NAA) and were involved in hormone regulation. Exogenous ABA and MeJA treatments significantly upregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes in the early stage of color conversion and made grape berries quickly colored. Interestingly, GA treatment downregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes in the early color-change period, but significantly upregulated in the middle color-change and ripening stages, therefore GA mainly modulated grape berry coloring in the middle- and late-ripening stages. Furthermore, NAA treatment downregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes and delayed the peak expression of genes. Meanwhile, to further recognize the potential functions of VvmiR156b/c/d, the mature tomato transient trangenetic system was utilized in this work. Results showed that transient overexpression of VvmiR156b/c/d in tomato promoted fruit coloring and overexpression of VvSPL9 inhibited fruit coloration. Finally, a regulatory network of the VvmiR156b/c/d-VvSPL9 module responsive to hormones modulating anthocyanin synthesis was developed. In conclusion, VvmiR156b/c/d-mediated VvSPL9 participated in the formation of grape color in response to multi-hormone signals.

Demethylation alters transcriptome profiling of buds and leaves in 'Kyoho' grape.

BACKGROUND: Grape buds and leaves are directly associated with the physiology and metabolic activities of the plant, which is monitored by epigenetic modifications induced by environment and endogenous factors. Methylation is one of the epigenetic regulators that could be involved in DNA levels and affect gene expression in response to stimuli. Therefore, changes of gene expression profile in leaves and bud through inhibitors of DNA methylation provide a deep understanding of epigenetic effects in regulatory networks. RESULTS: In this study, we carried out a transcriptome analysis of 'Kyoho' buds and leaves under 5-azacytidine (5-azaC) exposure and screened a large number of differentially expressed genes (DEGs). GO and KEGG annotations showed that they are mainly involved in photosynthesis, flavonoid synthesis, glutathione metabolism, and other metabolic processes. Functional enrichment analysis also provided a holistic perspective on the transcriptome profile when 5-azaC bound to methyltransferase and induced demethylation. Enrichment analysis of transcription factors (TFs) also showed that the MYB, C2H2, and bHLH families are involved in the regulation of responsive genes under epigenetic changes. Furthermore, hormone-related genes have also undergone significant changes, especially gibberellin (GA) and abscisic acid (ABA)-related genes that responded to bud germination. We also used protein-protein interaction network to determine hub proteins in response to demethylation. CONCLUSIONS: These findings provide new insights into the establishment of molecular regulatory networks according to how methylation as an epigenetic modification alters transcriptome patterns in bud and leaves of grape.

Characterization of VvSPL18 and Its Expression in Response to Exogenous Hormones during Grape Berry Development and Ripening.

The sequence and structure of grape SBP-box-like18 (VvSPL18) were identified and characterized to explore its regulatory roles during grape berry development and ripening. Homologous conservation across diverse plant species was observed, and its potential function and modulated roles in grapes were investigated. The results showed that VvSPL18 has an ORF sequence of 1,137 bp, encodes 378 amino acids, and is located on chromosome 14 of grapevine. VvSPL18 has the closest relationship with its homolog in soybeans. The promoter of VvSPL18 contains cis-elements responsive to gibberellins (GA) and salicylic acid (SA), indicating that this gene might respond to these hormones involved in the modulation of grape berry. VvSPL18 is mainly distributed in the nucleus. Expression profiles showed that VvSPL18 is highly expressed only at the veraison stage of the grape berry and is slightly expressed in other phases. RNA-seq data also revealed that VvSPL18 might participate in the modulation of grape berry development and ripening. Treatment with diverse hormones demonstrated that abscisic acid (ABA) had almost no effect on its expression, whereas naphthalene acetic acid (NAA) significantly upregulated its expression at the veraison stage. We also found that VvSPL18 has a GA-responsive cis-element but no NAA-responsive cis-element. GA could promote the expression of VvSPL18 with a peak at an earlier stage than NAA, suggesting that VvSPL18 responds faster to GA than to NAA. This result indicates that VvSPL18 might modulate berry development at this phase through an ABA-independent pathway, and it might directly respond to GA, but indirectly to NAA. Our findings provide insights into the functions of VvSPL18 in mediating grape berry development and ripening.

Cooling effect of rivers on metropolitan Taipei using remote sensing.

This study applied remote sensing technology to analyze how rivers in the urban environment affect the surface temperature of their ambient areas. While surface meteorological stations can supply accurate data points in the city, remote sensing can provide such data in a two-dimensional (2-D) manner. The goal of this paper is to apply the remote sensing technique to further our understanding of the relationship between the surface temperature and rivers in urban areas. The 2-D surface temperature data was retrieved from Landsat-7 thermal infrared images, while data collected by Formosat-2 was used to categorize the land uses in the urban area. The land surface temperature distribution is simulated by a sigmoid function with nonlinear regression analysis. Combining the aforementioned data, the range of effect on the surface temperature from rivers can be derived. With the remote sensing data collected for the Taipei Metropolitan area, factors affecting the surface temperature were explored. It indicated that the effect on the developed area was less significant than on the ambient nature zone; moreover, the size of the buffer zone between the river and city, such as the wetlands or flood plain, was found to correlate with the affected distance of the river surface temperature.