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Transcriptomic Approaches for Elucidating the Genes Network
Page | 167 coconut (Fan et al., 2013), rice (Zhang et al., 2013), and sugarcane (Cardoso-Silva et al., 2014) for identification of novel genes. Earlier, Roche GS-FLX 454 was the most widely used platform for de novo transcriptome sequencing, due to its long read length in different organisms, for example, ginseng (Sun et al., 2010), A. thaliana (Wall et al., 2009), and maize (Vega-Arreguin et al., 2009). The Illumina transcriptome was mainly used for the sequencing of organisms whose genome was sequenced (Li et al., 2010). It was confirmed in due course of time that the relatively short reads can be effectively assembled by the Illumina transcriptome, or whole genome de novo sequencing and assembly with the advantage of paired-end sequencing (Maher et al., 2009).
Figure 1: Schematic representation of protocol used for the Next-Generation Sequencing using Illumina HiSeq.
Page | 168 Starch
Starch is the most significant form of carbon reserve in plantsin terms of the amount made, the universality of its distributionamong different plant species, and its commercial importance. It consists of different glucose polymers arranged into a threedimensional,semi-crystalline structure-the starch granule. Thebiosynthesis of starch involves not only the production of thecomposite glucans but also their arrangement into an organizedform within the starch granule. The formation of the starchgranule can be viewed as a simple model for the formationof ordered three-dimensional polysaccharide structuresin plants. Understanding the biochemical basis for the assemblyof the granule could provide a conceptual basis forunderstanding other higher order biosynthetic systems suchas cellulose biosynthesis. For example, one emerging concept is that structurewithin the granule itself may determine or influence the wayin which starch polymers are synthesized. Starch is synthesized in leaves during the day from photosyntheticallyfixed carbon and is mobilized at night. It is alsosynthesized transiently in other organs, such as meristemsand root cap cells, but its major site of accumulation is in storageorgans, including seeds, fruits, tubers, and storage roots.
Almost all structural studies have used starch from storageorgans because it is readily available and commercially important;we therefore focus on starch biosynthesis in storageorgans.
However, where aspects of transient biosynthesis areclearly different from long-term reserve synthesis, referenceis made to biosynthesis in non-storage tissues. Starch is synthesized in plastids, which in storage organscommitted primarily to starch production are called amyloplasts (Figure 2). These develop directly from proplastids and have littleinterna1 lamellar structure. Starch may also be synthesized inplastids that have other specialized functions, such as chloroplasts (photosynthetic carbon fixation), plastids of oilseed (fatty acid biosynthesis), and chromoplasts of roots such ascarrot (carotenoid biosynthesis).
Figure 2: Schematic representation of starch biosynthesis pathway operating in plants
Page | 169 The Biochemistry of Starch Biosynthesis
The biosynthetic steps required for starch biosynthesis arerelatively simple, involving three committed enzymes: ADPglucosepyrophosphorylase (ADPGPPase; EC 2.7.7.23), starchsynthase (SS; EC 2.4.1.21), and starch branching enzyme (SBE;EC 2.4.1.28). Amylose and amylopectin are synthesized from ADPglucose, which is synthesized from glucose-1- phosphate andATP in a reaction that is catalyzed by ADPGPPase and thatliberates pyrophosphate. This enzyme is active within theplastid, which means that its substrates, glucose-1-phosphateand ATP, must also be present in the plastid. In chloroplasts, ATP may be derived from photosynthesis, but in non-photosyntheticplastids, it must be specifically imported from the cytosol, probably by an ADP/ATP translocator. The glucose-1-phosphate canbe supplied by the reductive pentose phosphate pathway inchloroplasts via phosphoglucoisomerase and phosphoglucomutase. In non-photosynthetictissues, it may be imported directly from the cytosol or synthesized in the plastid from glucose6-phosphatevia the action of a plastidialphosphoglucomutase. The pyrophosphate produced by ADPGPPase is removedby inorganic alkaline pyrophosphatase, which is probably confinedto plastids in both photosynthetic and nonphotosynthetictissues. The removal of this plastidial pyrophosphate effectivelydisplaces the equilibrium of the ADPGPPase reactionin favor of ADPglucose synthesis (Figure 3).
Figure 3: Schematic of the metabolic flux and expression of genes involved in sucrose to starch pathway in developing wheat seed. Upper box legend indicates level of gene expression and lower box legend indicates the time or developmental stage as days post- anthesis.
Page | 170 Further, SS catalyzes the synthesisof α (1-4) linkage between the non-reducing end ofa preexisting glucan chain and the glucosyl moiety of ADPglucose, causing the release of ADP.
SSs can use both amyloseand amylopectin as substrates in vitro. How the initial primersfor the synthesis of glucan chains are produced in vivo is notknown. The α (1-6) branches in starch polymers are made by SBE, which hydrolyzes an (l-4) linkage within a chain and then catalyzesthe formation of an α (1-6) linkage between the reducingend of the “cut” glucan chain and another glucose residue, probably one from the hydrolyzedchain. SBEs show somespecificity for the length of the α (1-4) glucan chain that theywill use as a substrate.
Remodelling Starch Biosynthesis pathway
The genes associated with starch biosynthesis pathway has not been fully identified and characterised. With the advent of technology like NGS and gel-free proteomics, now it becomes easy to identify the respective transcripts and their proteins associated with SBP.
Even, the transcriptional regulation of the genes coding for these enzymes has not yet been fully explored. Transcriptional regulation may be a more important mechanism for long-term control of genes expression especially during caryopsis development (Table 1).
Table 1: Identification of novel transcripts and SNIPs based marker associated with fructose, starch and sucrose metabolism pathways in wheat using de novo transcriptomic approach.
Page | 171 Posttranslational regulation including phosphorylation, interaction with 14-3-3 regulatory proteins and posttranslational redox activation, appear to be essential regulatory mechanisms controlling starch biosynthesis by providing a rapid response to short-term environmental changes. The pathway in terms of synthesis and regulation has not been extensively studied. In our lab, we have executed whole transcriptome sequencing of contrasting wheat cvs. HD2985, HD2329, Raj3765 and BT-Schomburgkin a tissue specific manner at different stages of growth and development and under differential stress treatment. The transcriptome data generated from the developing endosperm tissue of contrasting wheat cvs. was analysed and characterised using different bioinformatics software’s. We identified ~87, 100, and 46 novel transcripts associated with fructose, starch and sucrose metabolism pathway which was further validated in our lab. We identified 12 putative soluble starch synthase genes using de novo transcriptomic approach and cloned five of them for further functional validation (Table 1). Similarly, we identified 4 AGPase, 2 SBE and 2 GBSS genes from contrasting wheat cvs. based on the information generated using transcriptomic approach. We also identified ~100000 SNIPs lying in differentially expressed genes associated with starch metabolism (Table 1). These are the potential resources to be utilized for the breeding program in order to develop a ‘climate-smart’
crop.
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