Regulation of Flavonoid Biosynthesis During Tomato Fruit Ripening - Research Paper Example

Title: Four transcription factors regulation of flavonoid biosynthesis during tomato fruit ripening: Non Ripening (NOR); Ripening Inhibitor (RIN); Colourless Non-Ripening (CNR); TDR4 gene. Introduction 1.1 Background and Concise conclusion Tomato (Solanum lycopersicum), a fruit of the Solanacea family is rich in its flavonoid contents. Flavonoids are polyphenolic compounds that are ubiquitous in nature and are categorized, according to chemical structure, into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins and chalcones. The flavonoids are thought to have potential beneficial effects on human health, like antiviral, anti-allergic, antiplatelet, anti-inflammatory, antitumor and antioxidant activities. For these reasons, the study of flavonoid pathway has garnered much attention from the Scientific community. 1 The role of flavonoids as the major red, blue, and purple pigments in plants has gained these secondary products a great deal of attention over the years. From the first description of acid and base effects on plant pigments by Robert Boyle in 1664 to the characterization of structural and regulatory genes in the late 20th century, a wealth of information has been collected on the structures, chemical activities, and biosynthesis of these compounds. 2 Tomato is considered the ideal model to study climacteric fruit ripening for a combination of scientific and agricultural reasons. The importance of tomato as an agricultural commodity has resulted in decades of public and private breeding efforts that have yielded numerous spontaneous and induced mutations, including many that affect fruit development and ripening. Simple diploid genetics, small genome size, short generation time, routine transformation technology, and availability of genetic and genomic resources, including mapping populations, mapped DNA markers. 3 Numerous single gene mutations that regulate fruit size, shape, development, and ripening combined with dramatic and readily quantifiable ripening phenotypes (ethylene, color index, carotenoids, softening) have enhanced the use of tomato as a model for climacteric ripening. 4 Genetic regulation process is referred to as a ‘cascade’ because the function of one gene triggers the functioning of the corresponding genes. That is a feedback mechanism takes place. This cascade is observed to be irreversible and unidirectional in most cases. For the sake of understanding, we may liken the cascade effect of gene functioning to the falling of a row of dominoes. In plants with fleshy fruits, a major focus has been the dissection of biochemical and genetic regulatory cascades controlling ripening using tomato as a model species. Transcription regulation factors involve many genes that produce different flavonoids. Of interest in this study are four transcription factors: Non Ripening (NOR), Ripening Inhibitor (RIN), Colourless Non-ripening (CNR) and TDR4 gene. We shall study how each of these factors has an effect on the synthesis of flavonoids. 1.2 Aims Basis for approaches taken 2.1 The pathway: flavonoid biosynthesis The flavonoid biosynthetic pathway has been comprehensively reviewed by Dooner & Robbins 1991, Koes et al. 1994, Holton & Cornish 1995, Mol et al. 1998, Weisshaar & Jenkins 1998, Winkel-Shirley 2001. Thus the studies of the flavonoid pathway range from classical genetic analysis of flower color inheritance patterns by Mendel, through the establishment of their chemical structures, to efforts to understand the factors involved in their biochemical synthesis (Bohm 1998). Basic knowledge of the flavonoid biosynthesis was gained from experimental studies using radio-labeled precursors at the end of 1950’s. The development of more sophisticated methods in analytical chemistry and enzymology, and later in gene technology, has produced a vast number of studies and detailed information of the flavonoid biosynthesis in several plant species. 5 Flavonoid biosynthesis starts with general phenylpropanoid metabolism and leads to the nine major subgroups: the colorless chalcones, aurones, isoflavonoids, flavones, flavonols, and flavandiols (gray boxes), and the anthocyanins, condensed tannins, and phlobaphene pigments. The first committed step is catalyzed by chalcone synthase (CHS), which uses malonyl CoA and 4-coumaroyl CoA as substrates. Enormous variety of end products arise through terminal modification by the addition of sugars as well as methyl, ferulate, and other groups. A large number of enzymes like cinnamate-4-hydroxylase (C4H), chalcone isomerase (CHI), chalcone reductase (CHR), chalcone synthase (CHS), 4-coumaroyl:CoA-ligase (4CL) are employed in the pathway.6 2.2 Four transcription factors Transcription in molecular biology is the copying of RNA from a DNA pattern to create a new RNA molecule. A transcription factor is a protein that binds DNA at specific sites where it can regulate transcription. Without transcription factors, the creation of new RNA from DNA cannot occur. Transcription factors can be activated or deactivated by other proteins. Transcription factors are primarily involved in the initiation stage of RNA transcription. They are the key to determining where the DNA chain becomes "unzipped," creating a single strand to which RNA can be bound while it's being built.7 The role of four main transcription factors involved in Flavin Biosynthesis pathway is as discussed below: 2.2.1 Non Ripening (NOR) The ripening of fruits is found to be dependent on certain chemical signals. In articifial conditions, ripening is induced by treatment with ethylene. Wounding the fruit also triggers a reaction that ripens the fruit abnormally in that particular area. Recent studies have concluded that NOR (as also the LeMads- RIN genes) participate in ethylene-independent signaling in tomato, following a pathway similar to the one followed in the wild-type fruits in nature. Thus, the action of these two factors is independent of the two mutations, i.e. presence of ethylene or wounding. Interestingly accumulation of LE-ACS2 mRNA was stimulated by ethylene treatment in rin fruit but not in nor fruit. This suggests that the signaling pathways of RIN and NOR affect the regulation of ripening-associated genes in distinct ways, at least with respect to the expression of LE-ACS2. 8 2.2.2 Ripening Inhibitor (RIN) The LeMads-RIN gene, known as RIN for short, is a tandem Madbox gene. MADS refers to a family of genes (the name is an acronym formed from the initials of the four original members of the family). More than 100 MADS-box sequences have been found in species of microbes, animals and plants, and most play important roles in developmental processes. Most prominent, the MADS-box genes in ßowering plant. 9 Hybrid tomatoes have a long shelf life because they are picked raw from the vines and they do not ripen fully till long. Thus, though the fruit reaches the market in perfect condition, consumers complain of a bland taste as opposed to the juicy wild variety. The discovery of RIN may help tomatoes to stay on the vine for a longer time, thus acquiring the natural nutrients which give the succulent, juicy tang to the fruit. 2.2.3 Colourless Non-Ripening (CNR) Research carried out by scientists A. J. Thompson et al, as published in the journal Plant Physiol in 1999 suggets that the Colorless non-ripening (Cnr) mutation in tomato results in mature fruits with colorless pericarp tissue showing an excessive loss of cell adhesion. This pleiotropic mutation is an important tool for investigating the biochemical and molecular basis of cell separation during ripening. Real-time PCR and biochemical analysis demonstrated that the expression and activity of a range of cell wall-degrading enzymes was altered in Cnr during both development and ripening. These enzymes included polygalacturonase, pectinesterase (PE), galactanase, and xyloglucan endotransglycosylase. In the case of PE, the protein product of the ripening-related isoform PE2 was not detected in the mutant. In contrast with wild type, Cnr fruits were rich in basic chitinase and peroxidase activity. A microarray and differential screen were used to profile the pattern of gene expression in wild-type and Cnr fruits. They revealed a picture of the gene expression in the mutant that was largely consistent with the real-time PCR and biochemical experiments. These experiments also demonstrated that the Cnr mutation had a profound effect on many aspects of ripening-related gene expression. This included a severe reduction in the expression of ripening-related genes in mature fruits and indications of premature expression of some of these genes in immature fruits. The program of gene expression in Cnr resembles to some degree that found in dehiscence or abscission zones. The researchers have speculated that there is a link between events controlling cell separation in tomato, a fleshy fruit, and those involved in the formation of dehiscence zones in dry fruits. 10 2.2.4 TDR4 Like NOR and RIN, TDR4 gene is also a gene of the MADS-box genes family. These genes are putative transcription factors involved in regulating numerous developmental processes, such as meristem and organ identity in inflorescences and in flowers. Recent reports indicate that they are involved in other processes than flower development such as the establishment of developing embryos, seed coat and ultimately in root and fruit development. A team of researchers from Argentina has identified seven tomato MADS-box genes that are highly expressed during the first steps of tomato fruit development of which TDR4 is also found to play a decisive role. According to comparisons of their deduced amino acid sequences, the MADS-box genes were classified into two groups: (1) already identified tomato MADS-box genes previously defined as flower identity genes (TAG1, TDR4 and TDR6) and (2) new tomato MADS-box genes (TAGL1, TAGL2, TAGL11 and TAGL12). With the exception of TAGL12, which is expressed near uniformly in every tissue, TDR4 and other genes show an induction during the tomato fruit development phase I (anthesis) and phase II, when active cell division occurs. In situ hybridization analyses show a specific expression pattern for each gene within the fruit and embryo sac tissues suggesting an important role in the establishment of tissue identity. Yeast two-hybrid analyses indicate that some of these proteins could potentially form dimers suggesting they could act together to accomplish their proposed role. 11 3. Methods 3.1 Microarray analysis of flavonoid biosynthesis Microarray analysis has revolutionized the field of molecular genetics and enabled a thorough understanding of the molecular mechanisms underlying normal and dysfunctional biological processes in a manner that was not feasible in earlier years. Gene expression profiling or microarray analysis has enabled the measurement of thousands of genes in a single RNA sample. A variety of microarray platforms have been developed, but the basic idea for each is simple: a glass slide or membrane is spotted or "arrayed" with DNA fragments or oligonucleotides that represent specific gene coding regions. Purified RNA is then fluorescently- or radioactively labeled and hybridized to the slide/membrane. In some cases, hybridization is done simultaneously with reference RNA to facilitate comparison of data across multiple experiments. After thorough washing, the raw data is obtained by laser scanning or autoradiographic imaging. 12 Microarray tests on the flavonoid biosynthetic pathway reveal the role of regulation factors and enzymes that are functional in differrent stages of ripening. In a study carried out by a team of scientists, a large tomato expressed sequence tag (EST) dataset (152 635 total) was analyzed to gain insights into differential gene expression among diverse plant tissues representing a range of developmental programs and biological responses. These ESTs were clustered and assembled to a total of 31 012 unique gene sequences. To better understand tomato gene expression at a plant system level and to identify differentially expressed and tissue-specific genes, the team developed and implemented a digital expression analysis protocol. By clustering genes according to their relative abundance in the various EST libraries, expression patterns of genes across various tissues were generated and genes with similar patterns were grouped. In addition, tissues themselves were clustered for relatedness based on relative gene expression as a means of validating the integrity of the EST data as representative of relative gene expression. Arabidopsis and grape EST collections were also characterized to facilitate cross-species comparisons where possible. Tomato fruit digital expression data was specifically compared with publicly available grape EST data to gain insight into molecular manifestation of ripening processes across diverse taxa and resulted in identification of common transcription factors not previously associated with ripening. 13 References: 1. Buhler, Donald and Miranda, Christobal, ‘Anti-oxidant activities of Flavonoids’ URL: http://lpi.oregonstate.edu/f-w00/flavonoid.html 2. 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