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Comparative transcriptome analysis to investigate the high starch accumulation of duckweed (Landoltia punctata) under nutrient starvation.

ABSTRACT: BACKGROUND:Duckweed can thrive on anthropogenic wastewater and produce tremendous biomass production. Due to its relatively high starch and low lignin percentage, duckweed is a good candidate for bioethanol fermentation. Previous studies have observed that water devoid of nutrients is good for starch accumulation, but its molecular mechanism remains unrevealed. RESULTS:This study globally analyzed the response to nutrient starvation in order to investigate the starch accumulation in duckweed (Landoltia punctata). L. punctata was transferred from nutrient-rich solution to distilled water and sampled at different time points. Physiological measurements demonstrated that the activity of ADP-glucose pyrophosphorylase, the key enzyme of starch synthesis, as well as the starch percentage in duckweed, increased continuously under nutrient starvation. Samples collected at 0 h, 2 h and 24 h time points respectively were used for comparative gene expression analysis using RNA-Seq. A comprehensive transcriptome, comprising of 74,797 contigs, was constructed by a de novo assembly of the RNA-Seq reads. Gene expression profiling results showed that the expression of some transcripts encoding key enzymes involved in starch biosynthesis was up-regulated, while the expression of transcripts encoding enzymes involved in starch consumption were down-regulated, the expression of some photosynthesis-related transcripts were down-regulated during the first 24 h, and the expression of some transporter transcripts were up-regulated within the first 2 h. Very interestingly, most transcripts encoding key enzymes involved in flavonoid biosynthesis were highly expressed regardless of starvation, while transcripts encoding laccase, the last rate-limiting enzyme of lignifications, exhibited very low expression abundance in all three samples. CONCLUSION:Our study provides a comprehensive expression profiling of L. punctata under nutrient starvation, which indicates that nutrient starvation down-regulated the global metabolic status, redirects metabolic flux of fixed CO2 into starch synthesis branch resulting in starch accumulation in L. punctata.

SUBMITTER: Tao X

PROVIDER: S-EPMC3654882 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Comparative transcriptome analysis to investigate the high starch accumulation of duckweed (Landoltia punctata) under nutrient starvation.

Tao Xiang X Fang Yang Y Xiao Yao Y Jin Yan-Ling YL Ma Xin-Rong XR Zhao Yun Y He Kai-Ze KZ Zhao Hai H Wang Hai-Yan HY

Biotechnology for biofuels 20130508 1

<h4>Background</h4>Duckweed can thrive on anthropogenic wastewater and produce tremendous biomass production. Due to its relatively high starch and low lignin percentage, duckweed is a good candidate for bioethanol fermentation. Previous studies have observed that water devoid of nutrients is good for starch accumulation, but its molecular mechanism remains unrevealed.<h4>Results</h4>This study globally analyzed the response to nutrient starvation in order to investigate the starch accumulation ...[more]

PMID: 23651472

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Project description:As the fastest growing plant, duckweed can thrive on anthropogenic wastewater. The purple-backed duckweed, Landoltia punctata, is rich in starch and flavonoids. However, the molecular biological basis of high flavonoid and low lignin content remains largely unknown, as does the best method to combine nutrients removed from sewage and the utilization value improvement of duckweed biomass.A combined omics study was performed to investigate the biosynthesis of flavonoid and the metabolic flux changes in L. punctata grown in different culture medium. Phenylalanine metabolism related transcripts were identified and carefully analyzed. Expression quantification results showed that most of the flavonoid biosynthetic transcripts were relatively highly expressed, while most lignin-related transcripts were poorly expressed or failed to be detected by iTRAQ based proteomic analyses. This explains why duckweed has a much lower lignin percentage and higher flavonoid content than most other plants. Growing in distilled water, expression of most flavonoid-related transcripts were increased, while most were decreased in uniconazole treated L. punctata (1/6?×?Hoagland?+?800 mg•L-1 uniconazole). When L. punctata was cultivated in full nutrient medium (1/6?×?Hoagland), more than half of these transcripts were increased, however others were suppressed. Metabolome results showed that a total of 20 flavonoid compounds were separated by HPLC in L. punctata grown in uniconazole and full nutrient medium. The quantities of all 20 compounds were decreased by uniconazole, while 11 were increased and 6 decreased when grown in full nutrient medium. Nutrient starvation resulted in an obvious purple accumulation on the underside of each frond.The high flavonoid and low lignin content of L. punctata appears to be predominantly caused by the flavonoid-directed metabolic flux. Nutrient starvation is the best option to obtain high starch and flavonoid accumulation simultaneously in a short time for biofuels fermentation and natural products isolation.

Project description:BackgroundDuckweed is considered a promising source of energy due to its high starch content and rapid growth rate. Starch accumulation in duckweed involves complex processes that depend on the balanced expression of genes controlled by various environmental and endogenous factors. Previous studies showed that nitrogen starvation induces a global stress response and results in the accumulation of starch in duckweed. However, relatively little is known about the mechanisms underlying the regulation of starch accumulation under conditions of nitrogen starvation.ResultsIn this study, we used next-generation sequencing technology to examine the transcriptome responses of Lemna aequinoctialis 6000 at three stages (0, 3, and 7 days) during nitrogen starvation in the presence of exogenously applied sucrose. Overall, 2522, 628, and 1832 differentially expressed unigenes (DEGs) were discovered for the treated and control samples. Clustering and enrichment analysis of DEGs revealed several biological processes occurring under nitrogen starvation. Genes involved in nitrogen metabolism showed the earliest responses to nitrogen starvation, whereas genes involved in carbohydrate biosynthesis were responded subsequently. The expression of genes encoding nitrate reductase, glutamine synthetase, and glutamate synthase was down-regulated under nitrogen starvation. The expression of unigenes encoding enzymes involved in gluconeogenesis was up-regulated, while the majority of unigenes involved in glycolysis were down-regulated. The metabolite results showed that more ADP-Glc was accumulated and lower levels of UDP-Glc were accumulated under nitrogen starvation, the activity of AGPase was significantly increased while the activity of UGPase was dramatically decreased. These changes in metabolite levels under nitrogen starvation are roughly consistent with the gene expression changes in the transcriptome.ConclusionsBased on these results, it can be concluded that the increase of ADP-glucose and starch contents under nitrogen starvation is a consequence of increased output from the gluconeogenesis and TCA pathways, accompanied with the reduction of lipids and pectin biosynthesis. The results provide novel insights into the underlying mechanisms of starch accumulation during nitrogen starvation, which provide a foundation for the improvement of advanced bioethanol production in duckweed.

Project description:BackgroundLandoltia punctata can be used as renewable and sustainable biofuel feedstock because it can quickly accumulate high starch levels. ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed step during starch biosynthesis in higher plants. The heterotetrameric structure of plant AGPases comprises pairs of large subunits (LSs) and small subunits (SSs). Although several studies have reported on the high starch accumulation capacity of duckweed, no study has explored the underlying molecular accumulation mechanisms and their linkage with AGPase. Therefore, this study focused on characterizing the roles of different L. punctate AGPases. Methodology. Expression patterns of LpAGPs were determined through comparative transcriptome analyses, followed by coexpressing their coding sequences in Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, and Nicotiana tabacum.ResultsComparative transcriptome analyses showed that there are five AGPase subunits encoding cDNAs in L. punctata (LpAGPS1, LpAGPS2, LpAGPL1, LpAGPL2, and LpAGPL3). Nutrient starvation (distilled water treatment) significantly upregulated the expression of LpAGPS1, LpAGPL2, and LpAGPL3. Coexpression of LpAGPSs and LpAGPLs in Escherichia coli generated six heterotetramers, but only four (LpAGPS1/LpAGPL3, LpAGPS2/LpAGPL1, LpAGPS2/LpAGPL2, and LpAGPS2/LpAGPL3) exhibited AGPase activities and displayed a brownish coloration upon exposure to iodine staining. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays validated the interactions between LpAGPS1/LpAGPL2, LpAGPS1/LpAGPL3, LpAGPS2/LpAGPL1, LpAGPS2/LpAGPL2, and LpAGPS2/LpAGPL3. All the five LpAGPs were fusion-expressed with hGFP in Arabidopsis protoplasts, and their green fluorescence signals were uniformly localized in the chloroplast, indicating that they are plastid proteins.ConclusionsThis study uncovered the cDNA sequences, structures, subunit interactions, expression patterns, and subcellular localization of AGPase. Collectively, these findings provide new insights into the molecular mechanism of fast starch accumulation in L. punctata.

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Comparative transcriptome analysis to investigate the high starch accumulation of duckweed (Landoltia punctata) under nutrient starvation.

Publications

Comparative transcriptome analysis to investigate the high starch accumulation of duckweed (Landoltia punctata) under nutrient starvation.

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