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Chapter 6 SUMMARY AND Conclusion
Self-reliance in food crops is a critical measure of prosperity for nations. It is more so for developing countries like India because of the burgeoning population and high levels of poverty. That translates into an urgency and responsibility to direct maximal efforts towards identification of increasing variety of cash crops that can be cultivated in wide variation of climatic conditions, is accessible to farmers near the poverty line and is yet nutritionally rich. It is in this specific context that finger millet (Eleusine coracana) has gained prominence as the crop species for feeding the masses in the South East Asian region countries and in other parts of the world as well. The exceptionally high nutraceutical value of the finger millet grain resides in its characteristic to accumulate high levels of calcium. Calcium (Ca2+) is a vital element and is both a nutrient and signalling molecule in cellular pathways. However, the role and mechanisms underlying cell, tissue and organ-specific distributions of Ca2+ in plants are poorly understood. The Ca2+ content in edible parts viz., fruits, tubers and seeds is reported to be low. Nonetheless, finger millet accumulates exceptionally high Ca2+ content especially in seeds. More intriguingly, wide variation is found in seed Ca2+ content across the various genotypes of finger millet. The volume of work presented in this thesis is an attempt to decipher the genetic and epigenetic basis of high levels of seed calcium accumulation in finger millet. The salient features of present investigation are summarized below:
In this study, a genome-wide comparative in silico analysis of the calcium transporter gene family of two crop species, rice and sorghum was done. Gene annotation, multiple sequence alignment, domain analysis of Ca2+ transporters, identification of upstream cis-acting elements, phylogenetic tree construction and syntenic mapping of the gene family were performed using several bioinformatics tools. In silico analysis has revealed the existence of 28 and 31 members of transporter genes encoding channel, ATPase and exchanger in genomes of sorghum and rice, respectively. Multiple sequence alignment of these transporter proteins of sorghum and rice showed substantial conservation. In all Ca2+ ATPases, alignment revealed one conserved DKTGTLT motif. Phylogenetic analysis further segregated these proteins into four clusters each representing calcium channel, IIA & IIB type calcium ATPases and calcium exchanger. In addition, most of the members belonging to the same cluster also share one or more conserved domains. Therefore, a majority of the Ca2+ transporters in rice are expected to be functional orthologs of the Ca2+ transporters in sorghum. Furthermore, all clusters contain members from the two different species, although rice is a C3 plant whereas sorghum has C4 metabolic pathway. The Ca2+ transporters of these two plants cluster together with respect to their orthology, which suggests that the structure and function of most of these genes might have remain conserved during evolutionary timescale. It is important to note that this is observed in spite of rice and sorghum different metabolic pathways (C3 and C4 respectively). Further, motif analysis revealed some conserved motifs between channel, ATPase and exchanger in rice and sorghum. The analysis of cis-regulatory elements for the predicted calcium transporters revealed that majorly, the putative functions of these genes were associated with gene regulation of seed storage proteins, abiotic and biotic stress, photoperiod, growth hormone and meristem. Although the function of calcium transporters in calcium accumulation in seed has not been reported yet, the findings reported in this work: presence of seed-specific motif in the upstream regions, strongly suggests that these proteins might actually be instrumental for calcium accumulation.
Characterization of these proteins at physiological, molecular and structural level might shed some light on their functionality. The cause of gene movement and the erosion of gene colinearity between rice and sorghum chromosomes are yet to be deciphered. Nonetheless, the advent of comparative genomics will provide insights for such changes in chromosomal organization. The identification of syntenic relationship between rice and sorghum reported in this present study showed that calcium transporters were not uniformly distributed but rather clustered on certain chromosomes. The in silico analyses presented here, of the predicted transporter gene family has provided significant clues for exploring the expression and function of these calcium transporters under different environmental conditions. These studies would further help in understanding the molecular basis of many agriculturally important traits such as calcium accumulation in developing grains and their roles in development and defence against biotic and abiotic stress agents. Furthermore, elucidating the diversity, organization and phylogeny of calcium transporter gene family in sorghum would facilitate future annotation of ion transporters within genomes of other cereals as well.
These calcium (Ca2+) transporters, like Ca2+ channels, Ca2+ ATPases, and Ca2+ exchangers, are thus instrumental for signalling and transport. However, the molecular interactions by which they orchestrate the accumulation of Ca2+ in grain ¬lling had not yet been investigated. Hence in the present study, rice genome-wide gene expression data analysis was done to identify the potential calcium transporter genes that may be responsible for the spatial accumulation of calcium during grain ¬lling of calcium in the developing grain. These datasets included tissue profiling using microarray as well as sequencing-based MPSS (Massively Parallel Signature Sequencing). In silico expression analyses were performed to identify Ca2+ transporters that predominantly express during the different developmental stages of Oryza sativa. The MPSS data revealed that there were seven Ca2+ transporter genes (i.e., three IIB-type Ca2+ ATPases [Os02g08018, Os04g51610, and Os12g39660], two IIA-type Ca2+ ATPases [Os03g17310 and Os03g52090], and two Ca2+ exchangers [Os01g37690 and Os011g05070]), that expressed during the seed development stages. On the other hand the Affymetrix microarray dataset revealed 9 calcium transporters expressed significantly during the seed developing stages. Five out of the nine calcium transporters comprising of 2 calcium exchangers, 2 ER- type Ca2 + ATPases, and 1 PM-type ATPase were found to express at significantly higher levels (90% confidence level; Z-score ≥1.64) during developing stages of seed. Further, 4 additional calcium transporter genes (2 exchangers and 2 PM-type ATPases) were identi¬ed at 80% con¬dence level (i.e., Z-score ≥1.28). Some of these transporters (1 exchanger, 2 ER-type, and 2 PM-type ATPases) were found to be expressed up till late in the developmental stage S5. Whereas 3 exchangers and 1 PM-type ATPase were found to be expressed highly in the initial stages (S1 and S2) of seed development, but declined in the subsequent stages. Hence a total of 13 seed-preferential unique calcium transporters (7 from massively parallel signature sequencing (MPSS) data analysis, and 9 from microarray analysis) were identi¬ed through meta analysis. Analysis of variance (ANOVA) revealed differential expression of the transporters across tissues, and principal component analysis (PCA) exhibited their seed-speci¬c distinctive expression pro¬le. Interestingly, Ca2+ exchanger genes are highly expressed in the initial stages, whereas some Ca2+ ATPases genes are highly expressed throughout seed development. Furthermore, analysis of the cis-elements located in the promoter region of the subset of 13 genes suggested that seed specific motif like GCN4, Skn1 play essential roles in regulating the expression of Ca2+ transporter genes during rice seed development. Based on these results and the available literature, a hypothetical model was developed for explaining the transport and tissue speci¬c distribution of calcium in developing cereal seeds. In this model, how and where these ATPases and exchangers express and function in transporting calcium into the developing grain is proposed. The model may be extrapolated to understand the mechanism behind the exceptionally high level of calcium accumulation seen in grains like ¬nger millet.
However, the proposed model was circumstantial and further experimental evidence like gain- or loss-of-function studies were required to validate the hypothesis. Keeping in mind the question of high calcium content, parallel studies on rice, sorghum and ¬nger millet were preformed to study the expression pattern of calcium transporters across different stages of seed through Q-RT PCR.Therefore complemented genes identified through meta-analysis with sequence homology and Real-time quantitative PCR to first postulate genes involved in calcium accumulation and then validate them in homologous species of rice, sorghum and as well as two genotypic variants of finger millet which differ in their calcium content. All identified 9 calcium transporters genes were examined in the four different stages of seeds in each plant by quantitative RT-PCR (qRT-PCR) and found that the microarray and qRT-PCR results were almost similar. The results indicated that these genes are likely to participate in the accumulation of calcium in seed development. Nevertheless, the functional portions of genes from these three crops retain sufficient conservation to be readily recognized. Therefore, the acquisition of gene sequences from rice will be extremely valuable for the identification of expression of calcium transporters from the genomes of sorghum, finger millet and other grasses.
Non-model plant species present an unexplored opportunity in furthering biological understanding as well as providing gains in nutritional content, especially when the relevant species are third world country crops. Finger millet is a nutritionally important crop characterized by high-calcium content in its seeds. As its genome is yet un-sequenced, leave alone the cellular mechanism of calcium accumulation, identification of the genes involved in the process presents a daunting challenge. In the absence of a completely sequenced genome such as finger millet transcriptomic resources for non-model species by next generation sequencing (NGS) have proven extremely useful for plant research and seed development process. Therefore expression analysis and RNA-seq analysis created a comprehensive view of the participation of calcium transporters families in accumulation of calcium in grain felling. To build a transcriptome resource and to detect with high-sensitivity the genes involved in grain filling, RNA-seq of the two genotypic variants of finger millet was performed by pooling RNA across various S1 to S4 developing spikes. Thus attest the seed specific expression pattern of nine calcium transporters differing across developmental stages in four crop species. Using the transcriptome resource from RNA-seq it was reported for the first time the assembled gene sequence for the nine calcium transporters. Recent advances in high-throughput RNA sequencing (RNA-seq) have enabled tremendous leaps forward in our understanding of finger millet transcriptomes. This was an important step in deciphering the cellular mechanism and our proposed model of calcium transport in seed grains would form a guiding post for further biochemical and genomic investigations.
Calcium present in the seed may not be in the element form bound but probably present in complexed form with other seed specific compounds. Calcium transporters are actively involved in the uptake and transport of calcium in the cells, while calcium binding proteins are involved in the storage of this calcium. Therefore, a study was carried out to identify the members of sequestering calcium binding proteins which were involved in calcium accumulation and storage in seed. For identification of calcium binding protein in RNA–Seq data of sorghum and rice in different developing stages of seed like seed 5 days, seed 10 days, embryo 25 days and endosperm 25 days were analyzed. Total 134 CBPs and 143 CBPs were found in rice and sorghum in seed developing stages (seed 5 days, seed 10 days, embryo 25 days and endosperm 25 days) through RNA-seq analysis. A large number of genes related to CBP were found to be expressed in the early and middle stages. Box plot comparisons showed that in rice and sorghum as well, have higher levels of expression of CBPs in the Seed 5 days stage. Seed 10 days after pollination (DAP) and endosperm 25 DAP were the least consistent tissues among the rice and sorghum, possibly due to differences in, seed morphology, and/or endosperm content.
The work presented in this study can be divided in two broad categories:
- Calcium transporter genes
- Calcium binding proteins
Using a two-pronged strategy of studying calcium transporters as well as binding proteins, the guiding motive has been to elucidate the mechanism of calcium uptake, translocation and accumulation in Poaceae. In this initiative finger millet has been chosen as the subject of investigation. The principal reason to do is the extremely high level of calcium accumulation in finger millet grains as compared to other cereals. That finger millet is a non-model organism was both a challenge and a motivating factor. Non-availability of the finger millet genome made me devise creative experimental and bioinformatic approaches. By bringing together methodologies such as comparative genomics, meta-analysis of publicly available expression resources, statistical measures, structural modelling and de novo assembly of the finger millet transcriptome, a synthesis for calcium related genes is presented. The primary understandings from the investigations carried out can be concluded as –
- Starting with rice and sorghum, a genome-wide analysis of calcium transporter gene families is presented. This formed the basis of my fundamental understanding of the genomic architecture for these gene families. It is also understood that due to significant sequence conservation of these gene families across species, non-model species like the finger millet could still be studied using conserved sequence information.
- The next step was to investigate the activity and preferential nature if any, for calcium transporters in the seed tissue. As rice has been extensively studied in the research community, publicly available tissue expression profiling datasets were utilized to dissect out the preference of calcium transporters for the seed tissue. A set of 9 genes thus identified with seed-enriched expression pattern were used to construct a hypothetical model of calcium accumulation in the finger millet seed.
- The final step was to extend the scope of the study at by comprehensively detecting the candidate genes using finger millet transcriptome sequencing and subsequent de novo assembly. As an additional functional parameter, two genotypes of finger millet were investigated: Low calcium (LC) and High calcium (HC). Overcoming the computational challenge, It is able to demonstrate not only the detection of the candidate genes across the genotypes but as well as an increased activity of CaEx1 in the HC genotype.
- Consequent validations using real-time quantitative PCR were in concordance with the observations and findings from the de novo transcriptome assembly. Further using qRT-PCR it is seen that whereas some calcium transporter genes were expressed in the early stages of seed development, there were rest which were expressed throughout the satges.
- The study of CBPs provide a glimpse into the dynamic nature of the Poaceae genome and transcriptome, revealing players and mechanisms that may be responsible for evolutionary novelty and calcium accumulation.
Finger millet is a C4 metabolising plant and hence has special characteristics in comparison to rice. How the carbon metabolism cycle affects the nutraceutical content of seed grains is not well understood yet and forms a major point for future investigations. The nucleotide and protein sequence based analyses carried out extensively in this study, such as phylogenetics, motif search, domain studies and sequencing-based transcriptomics, advocate for more in-depth investigations into calcium related genes using sequence level information. The first ever attempted de novo transcriptome assembly is just the beginning towards a more wholesome and functional understanding of the finger millet genome and its cellular physiology. The insights gained from this assembly and the genes identified should pave the path for elucidation of further mechanisms of nutrient filling and seed biology.
Additionally, a transcriptome wide screening of calcium related genes in finger millet at the resolution of different seed developmental stages has not been attempted yet. In the pilot study we pooled RNA across developmental stages to maximize the chances of discovering novel calcium related genes. Nonetheless, a study with stage-wise resolution would be much more insightful and would tell unambiguously whether there is/ are a certain class(es) of genes involved throughout or there stage-specific classes of genes that show activity. Such a combined approach of using in silico analyses and experimental follow-ups will lead to comprehensive understanding of the crucial events of seed development in cereal grains. Identification of potential candidate genes/ protein domains will be formative for creating ‘smart’ crops enriched with high nutritional value (viz. Calcium) and also for the development of nutraceuticals in the form of value-added functional foods. Such fortified foods might also facilitate the calcium absorption through human intestine.
The emphasis on food security could not be more as India is poised to take over China as the country with the highest population. This could potentially lead to millions of more impoverished individuals and further higher rates of malnutrition. Crops like finger millet provide sustainable hope for increasing food security. More importantly, high nutraceutical content of finger millet grains could be a powerful antidote for combating malnutrition in underprivileged farmers of this country. Scientific efforts as detailed herein will be useful for eliminating not only hidden/ non-apparent malnutrition caused by deficiency of trace nutrients like calcium, but would also prevent health ailments arising due to nutrient deficiency.