High flavonoid accompanied with high starch accumulation triggered by nutrient starvation in bioenergy crop duckweed (Landoltia punctata)

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Combined omics; Duckweed; Flavonoids; Nutrient starvation; Starch; Uniconazole.

Fig. 1

Phenylalanine metabolism networks in L. punctata. The abbreviations correspond to enzymes involved in phenylalanine metabolic networks. Different colors represent different expression levels. PAL: phenylalanine ammonialyase, EC: 4.3.1.24. C4H: cinnamate 4-hydroxylase, EC: 1.14.13.11. 4CL: 4-coumarate-CoA ligase, EC: 6.2.1.12. HCT: hydroxycinnamoyl transferase, EC: 2.3.1.133. C3H: 4-coumarate 3-hydroxylase, EC: 1.14.14.9. CCoAOMT: caffeoyl-CoA O-methyl transferase, EC: 2.1.1.104. COMT: caffeic acid o-methyl transferase, EC: 2.1.1.68. F5H: ferulate 5-hydroxylase, EC:1.14.-.-. CCR: cinnamoyl-CoA reductase, EC: 1.2.1.44; CAD: cinnamyl-alcohol dehydrogenase, EC: 1.1.1.195; LACC: laccase, EC: 1.10. 3.2. CHS: chalcone synthase, EC: 2.3.1.74. CHI: chalcone isomerase, EC: 5.5.1.6. F3H: flavanone 3-hydroxylase, EC: 1.14.11.9. FLS: flavonol synthase EC:1.14.11.23. DFR: dihydroflavonol 4-reductase, EC: 1.1.1.234. F3’H: flavonoid 3'-hydroxylase, EC: 1.14.13.21. F3’5’H: EC: 1.14.13.88. FNS: flavone synthase, EC:1.14.11.22. ANS: anthocyanidin synthase, EC: 1.14.11.19. ANR: anthocyanidin reductase, EC:1.3.1.77. LAR: leucoanthocyanidin reductase, EC:1.17.1.3. AS1: aureusidin synthase, EC:1.21.3.6. The bold arrows show the main metabolic flux

Fig. 2

Expression changes of transcripts related to flavonoid biosynthesis based on RNA-Seq. A heatmap was drawn by HemI toolkit using log2FC values [75]. Most abbreviations correspond to the enzymes listed in Fig. 1. Transcripts with extremely low expression levels are not shown in this figure

Fig. 3

Expression changes of proteins involved in flavonoid biosynthesis based on iTRAQ. A heatmap was drawn by HemI toolkit according to log2FC values [75]. Most abbreviations correspond to the enzymes listed in Fig. 1

Fig. 4

Flavonoid profiles of uniconazole or full nutrient treated L. punctata. a flavonoids of uniconazole treated L. punctata, 0342014-5-11, 0242014-5-11 and 0322014-5-11 corresponded to UT-0, UT-72 and UT-240, respectively. b flavonoids of full nutrient treated L. punctata, 0252014-5-10 and 0332014-5-11 corresponded to FN-72 and FN-240, respectively

Fig. 5

Color change of frond underside under different growth conditions. L. punctata 0202 monoclonal was cultivated in 1/6 × Hoagland (FN), 1/6 × Hoagland and sprayed with 800 mg•L^-1 uniconazole (Aoke Biotech Corp, Japan) solution on the surface (UT), or distilled water for 12 days (NS)

Fig. 6

Growth status of L. punctata under different culture conditions. L. punctata 0202 monoclonal was cultivated in 1/6 × Hoagland, 1/6 × Hoagland spraying with 800 mg•L^-1 uniconazole (Aoke Biotech Corp, Japan) solution on the surface, or distilled water for 12 days