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.

Figure 1

Component, photosynthesis and respiration of nutrient starvation treated L. punctata and activity of ADP-glucose pyrophosphorylase. Fronds were collected at different time point and used for fresh weight, dry weight, protein and starch percentage analysis, and enzymatic activity assay for ADP-glucose pyrophosphorylase (EC: 2.7.7.27; AGP) respectively. The protein and starch percentage were calculated basing on dry weight. Activities of AGP were measured following the introduction of Nakamura, Y. et al. [52]. Photosynthesis rate and respiration rate measured at 0 h time point were defined as 100%. For each time point, three culture flasks were chose as replicates for these analyses. A: fresh weight and dry weight correspond to left Y-axis, protein content and starch content correspond to right Y-axis. B: AGP activities correspond to left Y-axis, photosynthesis and respiration correspond to right Y-axis.

Figure 2

Annotation rate and long-CDS containing sequences proportion. 74,797 contigs were used for BLASTX search. The X-axis represents the range of the contig length. Size distributions of the final assembled contigs (black) and number of long-CDS containing contigs (white) shown in vertical histograms correspond to left Y-axis. The percentage of BLASTX hits to size-grouped contigs shown in diamond corresponds to right Y-axis.

Figure 3

Differences between each pair of samples. Overlap examinations were performed basing on the resulting gene lists of three comparisons by VENNY [58]. Overlap among three groups, 2 h vs 0 h (blue), 24 h vs 0 h (yellow) and 24 h vs 2 h (green), were showed here.

Figure 4

Expression patterns of some key enzymes involved in photosynthesis and respiration. RbcS1, RbcS2, RbcS3 were RuBisCO small subunits, corresponding to transcripts comp34400_c0_seq1, comp34400_c0_seq2, comp34400_c0_seq3, respectively. RbcL1 and RbcL2 were RuBisCO large subunits, corresponding to comp17879_c0_seq1 and comp31501_c0_seq6. RCA1 and RCA2 were rubisco activases, corresponding to comp23075_c0_seq1 and comp23075_c0_seq2. TF1-A1, TF1-A2, TF1-A3 and TF1-A4 were ATPase alpha subunits, corresponding to comp26885_c0_seq1, comp26885_c0_seq2, comp26885_c0_seq3 and comp31538_c0_seq1. TF1-B was ATPase beta subunit, corresponding to comp34876_c0_seq1.

Figure 5

Expression patterns of some carbon metabolism related transcripts. Expression variations of some carbon metabolism related transcripts are displayed in the simplified starch and sucrose metabolism pathway. Red boxes indicate the up-regulated enzymes in response to nutrient starvation, green for down-regulated, gray means no significant difference was observed and white means this enzyme was not found in this study. The numbers in the upper half of the boxes correspond to the EC numbers, the numbers in the lower half, separated by slash, correspond to the expression levels of these enzymes shown in FPKM at 0 h, 2 h and 24 h respectively. 2.7.7.27: ADP-glucose pyrophosphorylase; 2.4.1.242: granule bound starch synthase; 2.4.1.1: glycogen phosphorylase; 2.4.1.21: soluble starch synthase; 3.6.1.21: adp-sugar diphosphatase; 2.4.1.18: starch branching enzyme; 3.2.1.1: alpha-amylase; 3.2.1.2: beta-amylase; 2.7.7.9: UDP-glucose pyrophosphorylase; 2.4.1.12: cellulose synthase; 2.4.1.13: sucrose synthase; 2.4.1.14: sucrose phosphate synthase; 3.1.3.24: sucrose-6-phosphate phosphatase; 3.2.1.20: alpha-glucosidase; 2.4.1.15: trehalose-6-phosphate synthase; 3.1.3.12: trehalose 6-phosphate phosphatase; 3.2.1.28: trehalase; 5.4.2.6: beta-phosphoglucomutase.

Figure 6

Expression patterns of lignin and flavonoid biosynthesis related transcripts. The abbreviations in the upper half of the box correspond to the enzymes involved in lignin and flavonoid biosynthesis, the numbers in the lower half, separated by slash, correspond to the expression levels shown in FPKM at 0 h, 2 h and 24 h respectively. Different colors mean different expression levels. PAL: phenylalanine ammonia-lyase, EC: 4.1. 1.5. C4H: cinnamate 4-hydroxylase, EC: 1.14.13.11. 4CL: 4-hydroxycinnamoyl-CoA ligase, EC: 6.2.1.12. 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. 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.