A genomics approach reveals that aroma production in apple is controlled by ethylene predominantly at the final step in each biosynthetic pathway
TL;DR: This analysis suggests that the initial and final enzymatic steps with the biosynthetic pathways are important transcriptional regulation points for aroma production in apple.
Abstract: Ethylene is the major effector of ripening in many fleshy fruits. In apples (Malus x domestica) the addition of ethylene causes a climacteric burst of respiration, an increase in aroma, and softening of the flesh. We have generated a transgenic line of 'Royal Gala' apple that produces no detectable levels of ethylene using antisense ACC OXIDASE, resulting in apples with no ethylene-induced ripening attributes. In response to external ethylene these antisense fruits undergo a normal climacteric burst and produced increasing concentrations of ester, polypropanoid, and terpene volatile compounds over an 8-d period. A total of 186 candidate genes that might be involved in the production of these compounds were mined from expressed sequence tags databases and full sequence obtained. Expression patterns of 179 of these were assessed using a 15,720 oligonucleotide apple microarray. Based on sequence similarity and gene expression patterns we identified 17 candidate genes that are likely to be ethylene control points for aroma production in apple. While many of the biosynthetic steps in these pathways were represented by gene families containing two or more genes, expression patterns revealed that only a single member is typically regulated by ethylene. Only certain points within the aroma biosynthesis pathways were regulated by ethylene. Often the first step, and in all pathways the last steps, contained enzymes that were ethylene regulated. This analysis suggests that the initial and final enzymatic steps with the biosynthetic pathways are important transcriptional regulation points for aroma production in apple.
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TL;DR: The new knowledge on their relative roles during tomato fruit development is evaluated with a view to understand their mechanism of action in fleshy fruits and it is envisaged that such detailed knowledge will help design new strategies for effective manipulation of fruit ripening.
Abstract: Plant hormones have been extensively studied for their roles in the regulation of various aspects of plant development. However, in the last decade important new insights have been made into their action during development and ripening, in both dry and fleshy fruits. Emerging evidence suggests that relative functions of plant hormones are not restricted to a particular stage, and a complex network of more than one plant hormone is involved in controlling various aspects of fruit development. Though some areas are extensively covered, considerable gaps in our knowledge and understanding still exist in the control of hormonal networks and crosstalk between different hormones during fruit expansion, maturation, and various other aspects of ripening. Here, we evaluate the new knowledge on their relative roles during tomato fruit development with a view to understand their mechanism of action in fleshy fruits. For a better understanding, pertinent evidences available on hormonal crosstalk during fruit development in other species are also discussed. We envisage that such detailed knowledge will help design new strategies for effective manipulation of fruit ripening.
405 citations
TL;DR: During this period there is a major switch in relative hormone levels of the fruit, involving an overall decrease in auxin, gibberellin, and cytokinin and a simultaneous increase in abscisic acid and ethylene.
Abstract: Plant species that bear fruit often utilise expansion of an ovary (carpel) or accessory tissue as a vehicle for seed dispersal. While the seed(s) develop, the tissue(s) of the fruit follow a common progression of cell division and cell expansion, promoting growth of the fruit. Once the seed is fully developed, the fruit matures and the surrounding tissue either dries or ripens promoting the dissemination of the seed. As with many developmental processes in plants, plant hormones play an important role in the synchronisation of signals between the developing seed and its surrounding fruit tissue(s), regulating each phase of fruit development. Following pollination, fruit set is achieved through a de-repression of growth and an activation of cell division via the action of auxin and/or cytokinin and/or gibberellin. Following fruit set, growth of the fruit is facilitated through a relatively poorly studied period of cell expansion and endoreduplication that is likely regulated by similar hormones as in fruit set. Once the seeds reach maturity, fruit become ready to undergo ripening and during this period there is a major switch in relative hormone levels of the fruit, involving an overall decrease in auxin, gibberellin and cytokinin and a simultaneous increase in abscisic acid and ethylene. While the role of hormones in fruit set and ripening is well documented, the knowledge of the roles of other hormones during growth, maturation and some individual ripening components is sketchy.
343 citations
Cites background from "A genomics approach reveals that ar..."
...Consistent with this, there are a significant number of publications linking the production of aroma with ethylene (Flores et al., 2002; Botondi et al., 2003; Defilippi et al., 2005; Schaffer et al., 2007)....
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TL;DR: Various developments that have taken place in the last decade with respect to identifying and altering the function of ripening-related genes have been described and role of ethylene and ethylene-responsive genes in ripening of fleshy fruit is included.
285 citations
Cites background from "A genomics approach reveals that ar..."
...Schaffer et al. (2007) identified 17 candidate genes that were likely to be the control points for ethylene with respect to aroma production....
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TL;DR: This report serves as a synopsis of the resources and initiatives of the Rosaceae community, recent developments in Rosaceae genomics, and plans to apply newly accumulated knowledge and resources toward breeding and crop improvement.
Abstract: The plant family Rosaceae consists of over 100 genera and 3,000 species that include many important fruit, nut, ornamental, and wood crops. Members of this family provide high-value nutritional foods and contribute desirable aesthetic and industrial products. Most rosaceous crops have been enhanced by human intervention through sexual hybridization, asexual propagation, and genetic improvement since ancient times, 4,000 to 5,000 B.C. Modern breeding programs have contributed to the selection and release of numerous cultivars having significant economic impact on the U.S. and world markets. In recent years, the Rosaceae community, both in the United States and internationally, has benefited from newfound organization and collaboration that have hastened progress in developing genetic and genomic resources for representative crops such as apple (Malus spp.), peach (Prunus spp.), and strawberry (Fragaria spp.). These resources, including expressed sequence tags, bacterial artificial chromosome libraries, physical and genetic maps, and molecular markers, combined with genetic transformation protocols and bioinformatics tools, have rendered various rosaceous crops highly amenable to comparative and functional genomics studies. This report serves as a synopsis of the resources and initiatives of the Rosaceae community, recent developments in Rosaceae genomics, and plans to apply newly accumulated knowledge and resources toward breeding and crop improvement.
282 citations
Cites background from "A genomics approach reveals that ar..."
...…ESTs from apple has identified 10 distinct sequences that can be classified as representatives of seven conserved plant microRNA (miRNA) families, and secondary structure predictions reveal that these sequences possess characteristic fold-back structures of precursor miRNAs (Schaffer et al., 2007)....
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...Teamed with transgenic plants, custom apple arrays have also revealed that ethylene controls substantial production of flavorassociated volatiles (Schaffer et al., 2007), and other studies have investigated early fruit development (Newcomb et al., 2006; Lee et al., 2007)....
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TL;DR: In this article, an open-pollinated seedling of cv Sangrado that has green foliage and develops red flesh in the fruit cortex late in maturity was identified as a duplication event that occurred during the evolution of apple within the Maloideae family.
Abstract: Anthocyanin accumulation is coordinated in plants by a number of conserved transcription factors. In apple (Malus × domestica), an R2R3 MYB transcription factor has been shown to control fruit flesh and foliage anthocyanin pigmentation (MYB10) and fruit skin color (MYB1). However, the pattern of expression and allelic variation at these loci does not explain all anthocyanin-related apple phenotypes. One such example is an open-pollinated seedling of cv Sangrado that has green foliage and develops red flesh in the fruit cortex late in maturity. We used methods that combine plant breeding, molecular biology, and genomics to identify duplicated MYB transcription factors that could control this phenotype. We then demonstrated that the red-flesh cortex phenotype is associated with enhanced expression of MYB110a, a paralog of MYB10. Functional characterization of MYB110a showed that it was able to up-regulate anthocyanin biosynthesis in tobacco (Nicotiana tabacum). The chromosomal location of MYB110a is consistent with a whole-genome duplication event that occurred during the evolution of apple within the Maloideae family. Both MYB10 and MYB110a have conserved function in some cultivars, but they differ in their expression pattern and response to fruit maturity.
265 citations
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TL;DR: In this paper, a different approach to problems of multiple significance testing is presented, which calls for controlling the expected proportion of falsely rejected hypotheses -the false discovery rate, which is equivalent to the FWER when all hypotheses are true but is smaller otherwise.
Abstract: SUMMARY The common approach to the multiplicity problem calls for controlling the familywise error rate (FWER). This approach, though, has faults, and we point out a few. A different approach to problems of multiple significance testing is presented. It calls for controlling the expected proportion of falsely rejected hypotheses -the false discovery rate. This error rate is equivalent to the FWER when all hypotheses are true but is smaller otherwise. Therefore, in problems where the control of the false discovery rate rather than that of the FWER is desired, there is potential for a gain in power. A simple sequential Bonferronitype procedure is proved to control the false discovery rate for independent test statistics, and a simulation study shows that the gain in power is substantial. The use of the new procedure and the appropriateness of the criterion are illustrated with examples.
83,420 citations
"A genomics approach reveals that ar..." refers methods in this paper
...The number of significant differentially expressed genes was examined using a 0.05 threshold using a nonadaptive FDR control (Benjamini and Hochberg, 1995)....
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TL;DR: This study enters into the particular topics of the relative quantification in real-time RT-PCR of a target gene transcript in comparison to a reference gene transcript and presents a new mathematical model that needs no calibration curve.
Abstract: Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT–PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
30,462 citations
"A genomics approach reveals that ar..." refers methods in this paper
...Raw cycle threshold scores were converted to quantities representing relative expression levels using a modified comparative cycle threshold method (Pfaffl, 2001) and with correction for different amplification efficiencies (Ramakers et al., 2003)....
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TL;DR: The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which opens up the possibility of studying the biological relevance of small expression differences.
Abstract: Gene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem. We outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data. The normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.
18,261 citations
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...4 (Vandesompele et al., 2002)....
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TL;DR: It is shown that the first approach can lead to PCR efficiencies that vary over a 0.2 range, whereas the second approach may be off by 0.26, and proposed linear regression on the Log(fluorescence) per cycle number data as an assumption-free method to calculate starting concentrations of mRNAs and PCRefficiencies for each sample.
3,536 citations
"A genomics approach reveals that ar..." refers methods in this paper
...Raw cycle threshold scores were converted to quantities representing relative expression levels using a modified comparative cycle threshold method (Pfaffl, 2001) and with correction for different amplification efficiencies (Ramakers et al., 2003)....
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3,251 citations
"A genomics approach reveals that ar..." refers methods in this paper
...Following volatile detection, apple fruits were peeled and skin tissues and cortex tissues (excluding the core) for each time point were snap frozen in liquid nitrogen before storage at 280 C. Total RNA was extracted using a method to extract RNA from pine (Pinus taeda) needles (Chang et al., 1993)....
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