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

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

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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. ResultsA 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.
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R E S E A R C H A R T I C L E Open Access
High flavonoid accompanied with high
starch accumulation triggered by nutrient
starvation in bioenergy crop duckweed
(Landoltia punctata)
Xiang Tao
1,3
, Yang Fang
1,2,3
, Meng-Jun Huang
1,2,3,4
, Yao Xiao
1,3
, Yang Liu
1,2,3
, Xin-Rong Ma
1,3*
and Hai Zhao
1,3*
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 mgL
-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: Duckweed, Flavonoids, Starch, Combined omics, Nutrient starvation, Uniconazole
Background
Flavonoids, also known as vitamin P, constitute a vast
class of secondary metabolites widely distributed in plants,
which encompasses more than 10,000 structures [1]. They
have a low molecular weight and a general structure of
three rings, including two phenyl rings (A and B) and a
heterocyclic ring (C). With different substituent groups,
flavonoids can be divided into seven subgroups, includ-
ing chalcones, flavones, flavonols, flavandiols, anthocya-
nins, condensed tannins and aurones [2]. Some
specialized forms of flavonoids can be synthesized by
some plant species, such as the isoflavonoids [3] and 3-
deoxyanthocyanins [4, 5]. Different flavonoids usually
play various roles in plants by regulating several develop-
mental processes [610]. Furthermore, these secondary
metabolites are well characterized as defense compounds
and signaling molecules that can withstand a wide array of
environmental stresses in plants and diseases in humans
* Correspondence: maxr@cib.ac.cn;zhaohai@cib.ac.cn
Equal contributors
1
Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu,
Sichuan 610041, China
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Tao et al. BMC Genomics (2017) 18:166
DOI 10.1186/s12864-017-3559-z
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
[11, 12], due to their capacity to absorb ultraviolet (UV)
radiation, and inhibiting the generation of reactive oxygen
species (ROS) [1315]. Through their ability to inhibit
DNA gyrase, energy metabolism and cytoplasmic mem-
brane function, flavonoids possess antifungal, antiviral and
antibacterial activity [16, 17].
Duckweed (Lemnacecae family) is the smallest and
simplest flowering aquatic plant in the world, and its
growth highly adaptable across a broad range of climates
[18]. It has a long yearly production period with an al-
most exponential growth rate, producing biomass faster
than most other plants. It can thrive on eutrophic waste-
water, through its ability to remove nutrients from sew-
age [19] and large amounts of CO
2
from the atmosphere
[2022]. In warm seasons, duckweed can remove up to
85% of total Kjehldahl nitrogen (TKN) and 78% of total
phosphorous (TP) from sewage [22]. The value of duck-
weed as a test species for the registration of agrochemi-
cals has been discussed worldwide [23]. A previous
study indicated that this plant possesses negligible lignin
content [24]. Depending on the duckweed species and
the growing conditions, the starch content of duckweed
ranges from 3% to 75% [2527]. Furthermore, it has
been found that the purple-backed duckweed has high
flavonoid content in crude plant form [28, 29], while
only flavonoid-rich fractions of the most prevalent flavon-
oid sources, tartary buckwheat or ginkgo, can be used to
extract these kinds of flavonoids [3032]. Together with a
much higher biomass production, duckweed should be
a more promising flavonoid resource plant than tartary
buckwheat and gingko. These characteristics make
purple-backed duckweed a potential sustainable source
for bioenergy production [33, 34], animal feed [35] and
even human food [36]. A company from Israel has found
that duckweed can address the challenges of consumer
health concerns, rising health care costs and food security
issues by exploring the nutritional value, traditional con-
sumption in Southeast Asia, and commercialization possi-
bilities of duckweed [36]. However, a method to combine
nutrients remove from sewage and the utilization value im-
provement of duckweed biomass remains as yet unknown.
In a previous study, it was found that the total flavonoid
content of L. punctata increased from 4.51% to 5.56% fol-
lowing nutrient starvation for 168 h [28], accompanied by
high starch accumulation for bioethanol fermentation [25].
Spraying with 800 mg · L
-1
uniconazole is an alternative
method to accumulate high levels of starch [26, 27], but
whether it underwent the same physiological and molecu-
lar alteration remains unknown. In this study, the changes
of flavonoids in full nutrient, starvation and uniconazole
treated L. punctata groups were investigated and com-
pared by a combined omics study. This provided molecular
support for the simultaneous accumulation of high starch
and high flavonoid levels in this bio-resource plant.
Results
Comprehensive transcriptome construction for L. punctata
In order to construct a comprehensive transcriptome for
L. punctata, Illumina HiSeq 2000 paired-end (PE) reads
of nutrient starvation (distilled water, NS) [25] and unico-
nazole (1/6 × Hoagland + 800 mgL
-1
uniconazole, UT)
[26, 27] responsive transcriptomes, and also the full nutri-
ent (1/6 × Hoagland, FN) transcriptome, were pooled to-
gether and de novo assembled using Trinity (v2012-06-08)
[37]. All PE reads were deposited in Sequence Read
Archive database (SRA) under accession number of
PRJNA185389. A total of 543,912,936 PE 90 bp reads
were obtained from the three RNA-Seq groups, corre-
sponding to 48.95 Gbp in total (Table 1). Furthermore,
155,903 contigs with lengths 200 bp were assembled,
corresponding to a transcriptome size of 170.34 Mb.
The average length, N50 length and max length was
1093 bp, 2190 bp and 17,234 bp, respectively. Among
these contigs, 51,873 were longer than 1000 bp and
26,931 were longer than 2000 bp. The results from scan-
ning of the Open Reading Frames (ORFs) of all contigs
showed that there were 67,061 ORFs with lengths 600 bp
(from ATG to stop codon), and 37,797 ORFs with
lengths 900 bp.
All PE reads were used separately for short-read align-
ment for each sample through the perl script provided with
the Trinity package (v2012-06-08) [37]. The number of
aligned reads for each contig was counted and used for ex-
pression profiling. To normalize the bias introduced by the
sequencing library size and mRNA composition, edgeR
(the Empirical analysis of Digital Gene Expression in R)
[38] in the Trinity package (v2012-06-08) [37] was used to
make an effective library size for each sample and
normalize the number of aligned reads per transcript to
generate a FPKM (Fragments Per Kilobase of tran-
scripts per Million mapped fragments) value using the
RESM-based algorithm. It was found that the number
of expressed transcripts ranged from 36,950 to 60,854,
with only 20,776 transcripts expressed in all samples
(Table 1). Furthermore, the results showed that full nu-
trient conditions stimulated more transcript expression
compared to the other two experimental groups.
A BlastX sequence similarity search against the non-
redundant protein database (NR) of NCBI [http://
www.ncbi.nlm.nih.gov/] was conducted by a locally-
installed blast program to investigate functional annota-
tion of each contig. BlastX results were then uploaded
to the Blast2GO platform [39, 40] for annotation. A total
of 98,106 (62.9%) contigs (transcripts) had significant
BlastX hits. Of the 26,931 contigs 2000 bp in length,
26,273 were annotated, corresponding to an annota-
tion rate of 97.6%. For the 51,873 contigs 1000 bp,
the annotation rate was 93.0% (annotated 48,266). For
contigs 900 bp and 600 bp, this rate was 91.7%
Tao et al. BMC Genomics (2017) 18:166 Page 2 of 14
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(50,762 of 55,328) and 85.3% (59,595 of 69,878),
respectively.
Biosynthetic network of phenylalanine metabolism
To construct the biosynthetic network related to phenyl-
alanine metabolism, enzyme codes were extracted and
Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathways retrieved from the KEGG web server [http://
www.genome.jp/kegg/]. Transcripts were detected that
corresponded to almost all of the enzymes involved in
flavonoid and lignin biosynthesis, except for flavone
synthase, aureusidin synthase, flavanone 7-O-beta-
glucosyltransferase and flavanone 7-O-glucoside 2''-
O-beta-L-rhamnosyltransferase (Fig. 1, Additional file 1:
Table S1, Additional file 2: Table S2). Phenylalanine ammo-
nialyase (EC: 4.3.1.24, PAL), cinnamate 4-hydroxylase (EC:
1.14.13.11, C4H) and 4-hydroxycinnamoyl-CoA ligase (EC:
6.2.1.12, 4CL) are the universal factors involved in flavonoid
and lignin biosynthesis [41]. Transcripts encoding these en-
zymeswerehighlyexpressedwithabundancehigherthan
100 FPKM. Cinnamoyl-CoA reductase (EC: 1.2.1.44, CCR)
and hydroxycinnamoyl transferase (EC: 2.3.1.133, HCT)
catalyze the initial reactionintheligninbiosynthesis
branch. Almost all transcripts related to CCR and HCT
had expression levels lower than 20 FPKM. Conversely,
chalcone synthase (EC: 2.3.1.74, CHS) and chalcone isom-
erase (EC: 5.5.1.6, CHI), the enzymes that catalyze the first
two reactions of flavonoid biosynthesis branch, were more
highly expressed (Fig. 1). This possibly explains why duck-
weed has a much lower lignin percentage than most other
plants [24, 34, 42, 43]. As p-coumaroyl CoA is the product
of a 4CL or C4H catalyzed reaction, it can be converted
into isoliquiritigenin or naringenin chalcone, then catalyzed
by chalcone isomerase (EC: 5.5.1.6, CHI) to feed this prod-
uct into the isoflavonoid biosynthesis pathway. Expression
data showed that CHS and CHI were both highly expressed
at over 100 FPKM, which may indicate that a large amount
of p-coumaroyl CoA was directed into the isoflavonoid
biosynthesis pathway. Meanwhile, the expression of fla-
vanone 3-hydroxylase (EC: 1.14.11.9, F3H), dihydroflavo-
nol 4-reductase (EC: 1.1.1.234, DFR) and anthocyanidin
synthase (EC: 1.14.11.19, ANS), which direct the meta-
bolic flux into the anthocyanin biosynthesis branch, were
all higher than 100 FPKM. However, flavone synthase (EC:
1.14.11.22, FNS) was unable to be detected in this study.
Of course, this may be because there was no expression of
FNS.
RNA-Seq based flavonoid biosynthetic analyses of
nutrient starvation or uniconazole treated L. punctata
Expression patterns of genes involved in specific pathways
can affect the metabolic flux. All transcripts described
above were quantified by RNA-Seq analyses (Fig. 2,
Additional file 1: Table S1, Additional file 2: Table S2).
Culturing duckweed in distilled water, the highest expressed
PAL, comp39767_c0_seq1, showed no obvious change
(132.07, 140.13 and 113.28 FPKM). The other highly
expressed PAL, comp39767_c0_seq2, was increased in
NS-2 (29.62 FPKM) and NS-24 (39.36 FPKM) when
compared with that in NS-0 (17.70 FPKM). Expression
level of comp46865_c0_seq1, a C4H encoding transcripts,
was increased from 78.22 FPKM to 152.93 FPKM in 24 h.
The expression level of comp46833_c0_seq1, a 4CL en-
coding transcript, was increased 2.22 times in NS-24
(294.61 FPKM) compared with that in NS-0 (132.43
FPKM). Spraying with 800 mg · L
-1
uniconazole resulted
in a slight initial increase in the expression of the two
highly expressed PAL, comp39767_c0_seq1 and comp39767_
c0_seq2, followed by a slight decrease after 72 h. The highest
expressed C4H, comp46865_c0_seq1, was increased in
UT-72 (197.19 FPKM) and UT-240 (189.65 FPKM)
when compared with that in UT-0 (102.44 FPKM), UT-2
(105.64 FPKM) and UT-5 (108.16 FPKM). Moreover, two
other poorly expressed C4H, comp45597_c0_seq1 and
Table 1 RNA-Seq statistics for different duckweed samples
Sample name Clean read Clean bases (bp) Q20(%) GC(%) Expressed transcripts
NS-0 41,337,098 3,720,338,820 97.02 56.90 38,056
NS-2 38,628,052 3,476,524,680 97.00 57.20 36,950
NS-24 38,789,556 3,491,060,040 97.03 57.12 38,627
UT-0 48,315,010 4,348,350,900 98.55 55.18 42,319
UT-2 48,390,098 4,355,108,820 98.57 55.26 42,693
UT-5 48,623,932 4,376,153,880 98.60 55.10 45,081
UT-72 48,282,456 4,345,421,040 98.56 55.30 45,789
UT-240 48,248,454 4,342,360,860 98.55 55.50 43,254
FN-2 45,491,706 4,094,253,540 97.89 55.31 43,497
FN-5 45,962,348 4,136,611,320 97.94 54.92 45,028
FN-72 45,954,756 4,135,928,040 97.96 55.12 50,300
FN-240 45,889,470 4,130,052,300 97.94 54.92 65,854
Tao et al. BMC Genomics (2017) 18:166 Page 3 of 14
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comp45597_c1_seq2, were increased during the first
240 h in the uniconazole treated group. However, the
results also showed that the highest expressed 4CL en-
coding transcripts (comp46833_c0_seq1, comp46135_
c0_seq2) were obviously suppressed by uniconazole. In
addition, several other 4CL were also suppressed, in-
cluding comp44243_c0_seq10, comp44243_c0_seq9,
comp35216_c0_seq1 and comp35216_c1_seq1. 4-
coumaroyl-CoA produced by 4CL can be catalyzed by
HCT to produce 4-coumaroylshikimate or catalyzed by
CHS to be converted into naringenin chalcone, which
directs the substrate into lignin biosynthesis or flavonoid
biosynthesis. Enzymes involved in the flavonoid metabol-
ism branch were carefully analyzed. It was found that a
CHS encoding transcript (comp46654_c0_seq1) was in-
creased by starvation treatment from the original 401.10
FPKM to 500.20 FPKM at 2 h and 519.48 FPKM at
24 h. Similarly, comp46654_c0_seq1 showed a comparable
upward tendency in the uniconazole treated group.
However, its expression level was much lower than in
NS-0, NS-2 and NS-24. Furthermore, expression level
ofseveralCHI,FLS,F3H,F3H, ANS, ANR, LAR, IFR
and GT4 encoding transcripts were increased by nutrient
starvation. Conversely, the highest expressed DFR tran-
script (comp36170_c1_seq2) was suppressed (176.81,
118.47 and 36.75 FPKM in NS-0, NS-2 and NS-24),
while several other lowly expressed DFR transcripts were
increased (comp19960_c0_seq2, comp44884_c0_seq1,
comp44884_c0_seq2 and comp44884_c0_seq3). In the
uniconazole treated group, the highly expressed FLS,
comp48210_c0_seq1, was increased in the first 5 h, but
suppressed thereafter by uniconazole. Meanwhile,
three F3H encoding transcripts (comp43561_c0_seq1,
comp43561_c0_seq3, comp43561_c0_seq4) were also
suppressed by this plant growth regulator. Upon culti-
vation of L. punctata in full nutrient (1/6Hoagland)
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. F3H: flavonoid 3'-hydroxylase, EC: 1.14.13.21. F35H: 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
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solution, the number of increased transcripts was larger
than that of the suppressed transcripts. For example,
comp39767_c0_seq4, comp39767_c0_seq6, comp46865_
c0_seq1, comp44243_c0_seq10, comp44483_c0_seq2,
comp35216_c0_seq1, comp35216_c1_seq1, comp35307_
c1_seq1, comp46833_c0_seq1, comp18376_c0_seq1,
comp41458_c0_seq1, comp46286_c0_seq1, comp46286_
c0_seq2, comp19956_c0_seq1, comp43561_c0_seq6,
comp46246_c0_seq1 were increased in full nutrient condi-
tions. These results suggest that these three different treat-
ments may trigger different molecular responses.
iTRAQ based flavonoids biosynthetic analyses of L.
punctata treated with nutrient starvation or uniconazole
RNA-Seq study provides a global expression pattern to
reveal mRNA composition, but it cannot reveal infor-
mation about the proteome. As the newest developed
quantitative technology, iTRAQ is widely used for
proteome characterization. In this study, iTRAQ data
of previous studies [28] was re-analyzed using the tran-
scriptome described above as a reference database. The
abundance of the most detected flavonoid related pro-
teins, including the PAL, C4H, 4CL, CHS, CHI, F3H
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
Tao et al. BMC Genomics (2017) 18:166 Page 5 of 14
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and ANS (Fig. 3, Additional file 3: Table S3), was de-
tected to be improved in L. punctata when grown in
distilled water. CHS (comp46654_c0_seq1), the first
pivotal catalyzer for the flavonoid biosynthetic branch,
was increased 6.92 times at 2 h, and 4.37, 8.71 and
15.28 times at 5, 24 and 72 h, respectively, when com-
pared with that of 0 h. Conversely, the first key enzyme
of the other branch of the phenylalanine metabolic net-
works, the lignin biosynthetic branch, failed to be detected
in starvation treated samples. Although 25 laccase tran-
scripts were assembled by our de novo RNA-Seq study,
none of the proteins corresponding to these transcripts
were detected by iTRAQ. F3H, DFR and ANS are the en-
zymes directing metabolic flux into the anthocyanin bio-
synthesis branch. In starvation treated L. punctata,the
expression of F3H (comp47195_c0_seq1) was increased
by 1.14, 1.04, 1.42 and 3.73 times at 2, 5, 24 and 72 h, re-
spectively, when compared with that of 0 h, whereas DFR
(comp44884_c0_seq2) was increased by 0.83, 0.83, 0.97
and 1.32, and 1.33, 1.00, 1.39 and 3.13 for ANS
(comp18732_c0_seq1), respectively. These results may in-
dicate that the metabolic flux was regulated by nutrient
starvation to direct more substrates toward the anthocya-
nin biosynthesis branch. Spraying with uniconazole re-
sulted in the levels of most flavonoid related proteins
being decreased. Three universal factors of phenylalanine
metabolism, PAL (comp39767_c0_seq1), C4H (comp46865_
c0_seq1) and 4CL (comp35307_c0_seq1), were suppressed
immediately (at 2 h) by uniconazole. Although the ex-
pression of C4H was increased thereafter, the 4CL was
suppressed in all of the four UT samples. The other
4CL, comp46135_c0_seq2, was also suppressed at 72 h
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
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and 240 h. Furthermore, CHI (comp41458_c0_seq1),
IFR (comp46286_c0_seq1, comp46286_c0_seq2), COMT
(comp44590_c0_seq3, comp44487_c0_seq1), 3GT
(comp19783_c0_seq1, comp40167_c0_seq4), UF3GT
(comp45471_c0_seq2), and 5AT (anthocyanin 5-aromatic
acyltransferase, comp45766_c0_seq1) showed a downward
trend by the application of uniconazole.
Expression of lignin biosynthesis related genes
Lignin provides mechanical support for plant growth,
but is not necessary in duckweed, which floats on water
surfaces. It has been found that most of the lignin biosyn-
thesis related genes in L. punctata had lower expression
than that seen in the flavonoid biosynthesis pathway, and
the last key gene involved in lignin biosynthesis was only
poorly expressed [25]. These results were supported by an
iTRAQ study of the same test samples [28]. When treated
with 800 mg · L
-1
uniconazole, the first enzyme of the lig-
nin biosynthetic branch, HCT, had limited expression
(Additional file 1: Table S1, Additional file 2: Table S2).
The highest expression level recorded, for HCT, was 14.64
FPKM in UT-5. For the other enzymes in this pathway,
only three transcripts had expression levels 100
FPKM, including comp46970_c0_seq1 (CCoAOMT),
comp44590_c0_seq3 (COMT) and comp36380_c1_seq1
(F5H). 25 laccase (LACC, EC: 1.10.3.2) encoding tran-
scripts were assembled and all had lower expression
levels than 10.00 FPKM (Additional file 1: Table S1,
Additional file 2: Table S2). The highest expressed tran-
script exhibited an expression abundance of 2.97, 5.10,
6.76, 4.95 and 2.75 FPKM in five samples, while expres-
sion levels of the other transcripts were all lower than
1.5 FPKM. iTRAQ proteomics profiling results also
strongly supported this view. When treated with nutri-
ent starvation, almost all key enzymes involved in lignin
biosynthesis were not detected [28]. When exposed to
uniconazole and full nutrient medium, most of these
assembled transcripts were not detected either.
Expression of flavone, flavonol, isoflavonoid and
anthocyanin biosynthesis involved genes
The results described above suggest that the metabolic flux
may be primarily directed to the isoflavonoid or anthocya-
nin biosynthesis branches in L. punctata (Fig. 1). To verify
this, enzyme encoding genes involved in flavone, flavonol,
isoflavonoid and anthocyanin biosynthesis were carefully
analyzed. It was found that almost all genes involved in iso-
flavonoid biosynthesis, or the flavone and flavonol biosyn-
thesis pathway were not detected in the transcriptome.
Despite most anthocyanin biosynthesis related genes failing
to be identified, most detected transcripts were increased
by nutrient starvation and uniconazole (Additional file 1:
Table S1, Additional file 2: Table S2). Five UDP-glucose
flavonoid 3-O-glucosyltransferase (EC: 2.4.1.51, UF3GT)
encoding sequences, including comp44420_c0_seq1,
comp44420_c0_seq2, comp44420_c0_seq3, comp38450_
c0_seq2 and comp47570_c0_seq1, were identified. Four
of them (comp44420_c0_seq2, comp44420_c0_seq3,
comp38450_c0_seq2 and comp47570_c0_seq1) were in-
creased by nutrient starvation, while being slightly in-
creased by uniconazole treatment and suppressed by
full nutrient treatment. Moreover, expression levels of
two anthocyanidin 3-o-glucosyltransferase (GT1, EC:
2.4.1.115, comp46472_c0_seq3, comp46472_c0_seq14)
genes were also increased by the first two treatments
described above, but decreased by full nutrient treatment.
Whereas 5AT (comp19598_c1_seq1) was only increased
by uniconazole, anthocyanin 3-o-beta-glucosyltransferase
(3GT, 2.4.1.238, comp33618_c0_seq1) was increased by
starvation. These observations support the hypothesis that
the metabolic flux was mainly directed into the anthocya-
nin biosynthesis branch and not the others.
Flavonoid content of uniconazole and full nutrient
treated L. punctata
In a previous study, it was found that the total flavonoid
content of L. punctata increased from 4.51% to 5.56%
during nutrient starvation for 168 h, of which seven of
the 17 components showed an obvious increase [28].
Growing L. punctata under natural conditions, the same
number of flavonoid compounds was separated by spec-
troscopic, chemical and biochemical methods, and four
of these were identified as new flavonoids in duckweed
[29]. However, whether these 17 flavonoid compounds
are the same as those observed in the starvation or uni-
conazole treated L. punctata has not been verified. In
this study, flavonoids were extracted and characterized
from uniconazole treated L. punctata following the
protocol described in the study of Wang, et al. [29]. The
results showed that a total of 20 compounds were sepa-
rated, including the additional compounds 1, 9, 14, 15,
16, 17, 18, 19 and 20 that did not separate in the previ-
ous study (Fig. 4). In contrast to the starvation treated L.
punctata, all of the 20 compounds were decreased by
uniconazole treatment. In addition, several compounds
(compound 1, 3, 4, 5, 6, 7, 8, 9, 13, 14 and 17) were in-
creased and several were decreased (compound 11, 12,
15, 18, 19 and 20) by full nutrient treatment. The total
flavonoid content of entire plants changed from 2.83%
to 0.94% and 3.37% in uniconazole and full nutrient
treated L. punctata, respectively. Furthermore, it was
found that purple coloration accumulated on the frond
underside in starved L. punctata, whereas no obvious
changes were observed in the full nutrient group, and
only slight changes seen for uniconazole treatment
group (Fig. 5). These results suggest that anthocyanin
accumulation may be one of the main factors of flavon-
oid increase caused by nutrient starvation.
Tao et al. BMC Genomics (2017) 18:166 Page 7 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Fig. 4 Flavonoid profiles of uniconazole or full nutrient treated L. punctata.aflavonoids 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. bflavonoids of full nutrient treated L. punctata, 0252014-5-10 and 0332014-5-11
corresponded to FN-72 and FN-240, respectively
Tao et al. BMC Genomics (2017) 18:166 Page 8 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Discussion
Special phenylalanine metabolic flux resulted in high
flavonoid and low lignin content
Duckweeds are the fastest growing and smallest flower-
ing plants. A number of studies have shown the poten-
tial for duckweeds to be developed as feedstock, for
biofuel production and as a natural purifier for swine
wastewater treatment [21, 25, 4449], due to its high fer-
mentable starch level (40-70% of dry weight), negligible
lignin content and capacity to thrive on anthropogenic
wastewater [24, 25, 33, 42, 5052]. More recently, its
high flavonoid content (>4% of dry weight) in crude
plant form [28, 29], has been found. As flavonoids play a
crucial role in plant defense against pathogens [16, 53],
they can be used to partially explain why duckweeds are
rarely infected by pathogens. With near-exponential
growth rates, duckweed can achieve a biomass of 13 to
38 metric tons/hectare/year dry weight [54], resulting in
more than 520 kg/hectare/year flavonoid production.
However, the molecular mechanism responsible for high
flavonoid content remains largely uninvestigated. Newly-
developed, high-throughput DNA sequencing technol-
ogy provides an opportunity for genome-wide global
transcriptome studies and metabolic pathway analyses.
In this study, phenylalanine metabolism involved genes
were carefully analyzed based on the RNA-Seq data of
starvation, uniconazole and full nutrient treated L.
punctata. Except flavone synthase, aureusidin synthase,
flavanone 7-O-beta-glucosyltransferase, and flavanone
7-O-glucoside 2''-O-beta-L-rhamnosyltransferase, all of
the other key enzymes involved in phenylalanine
metabolism were successfully detected from the transcrip-
tome (Fig. 1, Additional file 2: Table S2). p-coumaroyl
CoA is the common substrate for the biosynthesis of fla-
vonoid and lignin. The expression levels of CHS, HCT
and CCR provided cues that p-coumaroyl CoA may be
predominantly directed into the flavonoid branch and
rarely into the lignin branch, resulting in the high flavon-
oid and low lignin content in L. punctata (Fig. 1). It is well
known that lignin primarily provides mechanical support
for plants to stand upright and enables xylems to with-
stand the negative pressure generated during water trans-
port. Consequently, lignin is useless for L. punctata as
these plants usually grow on the waters surface with no
need for mechanical support. To effectively avoid the ac-
cumulation of a helpless product, the metabolic flux is
therefore mainly directed into the flavonoid branch. With
this characteristic, L. punctata can be developed as a
promising resource plant for biofuels fermentation and
flavonoids extraction.
The following iTRAQ based proteomics analyses sup-
ported these results. The majority of lignin synthesis in-
volved transcripts identified by RNA-Seq were not
detected in the iTRAQ study. Although possibly due to
technology bias, these lignin related enzymes were present
in levels lower than the detection limit of this technology,
as most enzymes involved in the other branch were suc-
cessfully quantified using the same samples. Moreover, the
global expression pattern of the phenylalanine metabolism
pathway revealed that the metabolic flux was directed to
the following anthocyanin biosynthesis branch with prior-
ity, but not the isoflavonoid biosynthesis or flavone and
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 mgL
-1
uniconazole (Aoke Biotech Corp, Japan) solution on the surface (UT), or distilled water for 12 days (NS)
Tao et al. BMC Genomics (2017) 18:166 Page 9 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
flavonol biosynthesis branches (Fig. 1). Since L. punctata
also known as purple-backed duckweed due to the
reddish-purple tint on the underside of its fronds as a re-
sult of anthocyanin production, the metabolic flux can be
explained by its morphological characteristics. Further-
more, almost all of the enzymes involved in isoflavonoid
biosynthesis or the flavone and flavonol biosynthesis path-
way, failed to be de novo assembled using RNA-Seq reads,
probably because of extremely low levels of expression.
This study combined omics data to investigate flavonoid
biosynthesis in L. punctata for the first time. The expres-
sion profiling not only gives a valuable insight into the
molecular biological basis of the high flavonoid content in
L. punctata, but also supports the morphological charac-
teristics of this plant species by the analyses of metabolic
flux.
Nutrient starvation is the optimized method to accumulate
high starch and flavonoid content simultaneously in this
resource plant
When growing L. punctata in distilled water, almost all
essential mineral nutrientswere deficient resulting in
extreme nutrient starvation. To cope with this abiotic
stress, L. punctata immediately increased expression of
some transporters with the aim of increasing nutrient
acquisition [25], but without success due to the absence
of nutrients. The global physiological and metabolic sta-
tus was altered and starch biosynthesis was enhanced,
resulting in a high starch accumulation of 45% (dry
weight) in 168 h [25]. These effects may be explained as
a stress escape or stress avoidance response to complete
the life cycle in advance by storing most carbon nutrients
and energy in starch (Fig. 6) [55]. As a class of important
defense compounds, over-accumulation of flavonoids in
plants can enhance stress tolerances by inhibiting the gen-
eration of ROS in plants [1315, 5661]. Manipulating
flavonoid biosynthetic gene expression is an effective
method to alter the accumulation of flavonoids in Arabi-
dopsis and other plants [6265]. In nutrient starvation
treated L. punctata, transcriptome analyses showed that
most flavonoid involved transcripts were increased (Fig. 2,
Additional file 2: Table S2), which was confirmed by
iTRAQ based proteome results (Fig. 3, Additional file 3:
Table S3). Metabolomic studies revealed a flavonoid accu-
mulation from the original 4.51 to 5.56% (dry weight) after
168 h, with seven of the 17 detected flavonoid compounds
having increased significantly [28], possibly due to the al-
tered expression of flavonoid biosynthetic genes. Further-
more, purple color accumulation on the frond undersides
correlated with the levels of flavonoids (Fig. 5). Overall,
these integrated results from transcriptome, proteome,
metabolome and morphology reveal a flavonoid based
stress response in distilled water.
Uniconazole, a plant growth retardant, has been exten-
sively applied in plants to increase tolerance and im-
prove quality by regulating endogenous hormone levels
[6669]. Culturing duckweed in 1/6 × Hoagland medium
andsprayingwith800mg·L
-1
uniconazole is an optimized
method to accumulate high starch content for bioethanol
fermentation and biomass accumulation [26, 27]. The con-
tent of starch was increased from 3.16% to 48.01% in 240 h
[27]. Different from that in distilled water treated L. punc-
tata, the biomass of uniconazole treated L. punctata was
almost equal to the control (1/6 × Hoagland) (Fig. 6), indi-
cating that 1/6 × Hoagland and 800 mg · L
-1
uniconazole
did not create stress conditions and consequently did not
trigger extra demand for flavonoids. As expected, flavonoid
content was decreased from 2.83% to 0.94% at 168 h. Simi-
larly, expression profiling results showed that more than
half of the flavonoid involved genes were suppressed by
this growth retardant. In our previous study, it was found
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 mgL
-1
uniconazole (Aoke Biotech Corp,
Japan) solution on the surface, or distilled water for 12 days
Tao et al. BMC Genomics (2017) 18:166 Page 10 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
that uniconazole increased the content of abscisic acid
(ABA) and cytokinins (CK), and suppressed the synthesis
of gibberellin (GA) [26, 27]. As ABA, CK and GA usually
crosstalk with salicylic acid (SA), jasmonic acid (JA), other
endogenous hormones and small molecule regulators, al-
terations ion the levels of these regulators should affect the
regulatory network in L. punctata.Previousstudieshadre-
vealed that different endogenous hormones usually play
different roles in flavonoid biosynthesis. For example, su-
crose can induce anthocyanin biosynthesis, but its effect is
repressed by the addition of GA, whereas JA and ABA
have a synergic effect with sucrose [70]. Accordingly, the
decreased level of flavonoid may be a result of interference
to the whole hormonal regulatory network in uniconazole
treated L. punctata. However, whether the flavonoid de-
crease is primarily caused by the change of ABA, GA, CK,
or uniconazole directly affecting the expression of some
flavonoid related key genes, still requires further investiga-
tion in the future.
Full nutrient (1/6 × Hoagland) is an optimized culture
medium, which usually cannot provide abiotic stress. So
that the physiological status of L. punctata would not be
altered in this study, starch and flavonoid content were
kept at normal levels. Although the growth status of the
nutrient starvation group was obviously suppressed
(Fig. 6), the total starch weight was increased by 42
times in 7 days [25]. Comparatively, the biomass of uni-
conazole treated L. punctata was almost equal to the
control (1/6 × Hoagland), with starch weight increasing
by 46 times in 7 days [26]. Therefore, although starva-
tion limits the accumulation of biomass, it can still have
the same effect on starch accumulation, which is caused
by a much lower dry matter rate in uniconazole treated
L. punctata. Since the flavonoid content was increased
by nutrient starvation, it can be surmised that nutrient
starvation is the optimized method for obtaining high
starch and high flavonoid content simultaneously in L.
punctata, while uniconazole treatment can only produce
high starch content.
Although some sampling time points of the RNA-Seq,
iTRAQ and metabolome studies were inconsistent, the
combined omics data reflect the changing trends of
mRNAs, proteins and flavonoid compounds, as these high
throughput technologies can characterize global gene ex-
pression patterns and metabolic status. In addition, a few
discordant results appeared in the expression results in
this study. As most enzymes were encoded by more than
one transcript, the non-matching results may have been
due to functional redundancy and spatio-temporal expres-
sion specificity of enzyme encoding transcripts.
Conclusions
Transcriptome and iTRAQ based expression profiling
revealed that high flavonoid and low lignin content of L.
punctata resulted primarily from phenylalanine meta-
bolic flux directed towards the flavonoid biosynthetic
pathway. Together with the metabolome assays, it was
found that full nutrient medium generated high biomass
with low starch and stable flavonoid content, unicona-
zole only induced starch accumulation accompanied by
a decreased flavonoid content, while nutrient starvation
triggered the accumulation of starch and flavonoids sim-
ultaneously. L. punctata has the potential to be devel-
oped as a resource plant for biofuel fermentation and
flavonoid extraction.
Methods
Plant materials and treatments
Monoclonal L. punctata 0202 was cultivated in 1/6 ×
Hoagland nutrient solution (total N = 58.3 mg/L, P =
25.8 mg/L) for 14 days under a 16/8 h day/night cycle,
with a light intensity of 130 μmol/m
2
/s, and a
temperature of 25 °C/15 °C during the day/night. For
the nutrient starvation group (NS), fresh fronds were
transferred into distilled water for further cultivation
over a period of two weeks. For the uniconazole treated
group (UT), fronds were subsequently cultivated in 1/
6 × Hoagland solution and sprayed on the surface with
800 mg · L
-1
uniconazole (Aoke Biotech Corp, Japan)
solution. The other groups were cultivated in 1/6 ×
Hoagland solution (FN). Different time points following
the transfer of fronds into different media were selected
for flavonoid analyses. For each time point, more than
3 g fresh fronds were collected from three culture flasks
for each sample, corresponding to a total of >800
individuals.
RNA extraction and RNA-Sequencing analyses
For each frond sample, more than 1 g fronds was
ground into powder in liquid nitrogen. Total RNA was
extracted using OMEGAPlant DNA/RNA kit (OMEGA,
USA), following the manufacturers instructions, and gen-
omic DNA was removed by DNase I (Fermentas, USA).
More than 20 μg total RNA was then submitted to Beijing
Genomics Institute (BGI)-Shenzhen, Shenzhen, China
[http://www.genomics.cn] for quality control. The purity,
concentration and RNA integrity number (RIN) were
measured by an Agilent 2100 Bioanalyzer or SMA3000.
Qualified total RNAs were used for the following
mRNA purification and 200 bp fragmented cDNA library
construction, identical to that described in our previous
study [25].
The validated fragmented cDNA library was submitted
to the Illumina Hiseq 2000 platform at BGI for tran-
scriptome sequencing. The 90 bp paired-end (PE) read
sequence and base-calling quality values were obtained
following the manufacturers instructions. The raw PE
reads were qualified by removing the reads with adapter
Tao et al. BMC Genomics (2017) 18:166 Page 11 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
sequence or excessive unknown bases. The clean reads
from the different samples were then pooled together
and de novo assembled using Trinity (v2012-06-08) with
the default parameters [37]. Length distribution was
assessed by common perl scripts to generate the N50
number, average length, max length and contig number
during different length intervals.
To profile the genome-wide expression patterns, all reads
were aligned back to the assembly using perl scripts in the
Trinity package (v2012-06-08) [37] for each RNA-Seq sam-
ple separately. The aligned read number was calculated
and presented as digital expression levels for each contig.
These values were then normalized for each RNA-Seq
sample by RESM-based algorithm using perl scripts in the
Trinity package (v2012-06-08) [37] to get FPKM values.
A BlastX sequence similarity search against the non-
redundant protein database (NR) of NCBI [http://
www.ncbi.nlm.nih.gov/] was conducted by a locally installed
blast program (ncbi-blast-2.2.28+, ftp://ftp.ncbi.nlm.nih.gov/
blast/executables/blast+/) to investigate functional annota-
tion for each contig. BlastX results were uploaded into
the Blast2GO platform [39, 40] for Kyoto Encyclopedia
of Genes and Genomes (KEGG) and Gene Ontology
(GO) annotation.
Protein extraction and iTRAQ based proteomic analyses
For each frozen sample, total protein extraction, qualifi-
cation and digestion were performed as the method de-
scribed in our previous study [28]. The digested peptides
were labeled following the manufacturers protocol with
iTRAQ® Reagent 8-plex Kit (AB SCIEX, USA) and sub-
sequently used for LC-MS/MS analyses using an AB
SCIEX TripleTOF5600 mass spectrometer (AB SCIEX,
USA), coupled with an LC-20AB HPLC Pump system
(Shimadzu, Kyoto, Japan).
MS/MS data acquisition was performed with Analyst®QS2.0
software (AB SCIEX, USA), and processed by searching
against the database generated from the annotated tran-
scriptome using the ParagonAlgorithm and the Mascot
search engine (Matrix Science, London, UK; version
2.3.02). The relative abundance was analyzed by the report
ion peak areas as previously described [71]. For protein
quantitation, it was required that a protein contains at
least two unique peptides.
Flavonoid content and classification
Flavonoid extraction and isolation were performed follow-
ing the methods described in our previous study [29]. The
flavonoid content of each frond sample was measured by
spectrophotometry with a spectrophotometer (Varioskan
Flash, Thermo Corp, USA) and HPLC (Thermo spectra
system AS3000, Thermo Corp, USA)-UV (Thermo
UV6000 Detector, USA) following the methods [72, 73].
HPLC/MS analyses of flavonoids were performed on
an Agilent series 1100 HPLC instrument (Agilent,
Waldbronn, Germany) coupled with a quadrupole time-
of-flight (Q-TOF) mass spectrometry (micrOTOF-Q II;
Bruker, Bremen, Germany) mainly in positive-ion mode.
The ESI source conditions were set following the method
of Yang [74]. The mass data were processed by Bruker
Compass DataAnalysis 4.0 software.
Additional files
Additional file 1: Table S1. Sequence annotation and expression
profiling. (XLSX 20158 kb)
Additional file 2: Table S2. Transcript levels of flavonoid metabolism
related contigs. (XLSX 58 kb)
Additional file 3: Table S3. Quantities of flavonoid metabolism related
proteins. (XLSX 11 kb)
Abbreviations
ABA: Abscisic acid; CKs: Cytokinins; DW: Dry weight; EC: Enzyme codes;
FN: Full nutrient; FPKM: Fragments Per Kilobase of transcripts per Million
mapped fragments; GA: Gibberellins; iTRAQ: Isobaric tags for relative and
absolute quantitation; JA: Jasmonic acid; log2FC: log2 fold-change;
NGS: Next-generation sequencing; NR: Non-redundant protein database;
NS: Nutrient starvation; PE: Paired-end; RNA-Seq: RNA-sequencing;
SA: Salicylic acid; UT: Uniconazole treated
Acknowledgements
Not applicable.
Funding
This work was supported by the National Natural Science Foundation of China
(No. 31400218), the National Key Technology R&D Program of China (No.
2015BAD15B01) and the Projects of International Cooperation of Ministry
of Science and Technology of China (No. 2014DFA30680).
Availability of data and materials
The transcriptome datasets supporting the conclusions of this article are
available in the [NCBIs Sequence Read Archive database (SRA) database]
repository under the accession number of PRJNA185389 [unique persistent
identifier and hyperlink to dataset(s) in http://www.ncbi.nlm.nih.gov/
sra?term=PRJNA185389].
Authorscontributions
XT conceived the study, carried out the data analysis, drafted and revised the
manuscript. YF drafted and revised the manuscript. YX carried out the data
analysis and revised the manuscript. MJH carried out the biochemical assays
and HPLC experiment, drafted and revised the manuscript. YL carried out the
biochemical assays and revised the manuscript. XRM conceived the study
and revised the manuscript. HZ conceived the study and revised the manuscript.
All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
Not applicable.
Author details
1
Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu,
Sichuan 610041, China.
2
University of Chinese Academy of Sciences, Beijing
100049, China.
3
Key Laboratory of Environmental and Applied Microbiology,
Chinese Academy of Sciences, Chengdu 610041, China.
4
College of Life
Science & Forestry, Chongqing University of Art & Science, Yongchuan,
Chongqing 402160, China.
Tao et al. BMC Genomics (2017) 18:166 Page 12 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Received: 8 June 2016 Accepted: 7 February 2017
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... Their structure contains a diphenyl propane backbone (C6-C3-C6) where a benzene ring (A) is attached with a pyrone ring (C), which is also substituted by a phenyl ring at its 2 or 3 positions [26]. More than 10,000 flavonoids have been reported from plants and they are mostly grouped into major sub-classes namely flavanones, flavones, isoflavones, flavonols, flavanols, and anthocyanidins [27]. Similarly, hydroxycinnamic acids (HCAs) and their conjugates are the other predominant plant phytochemicals synthesized via the phenylpropanoid pathway. ...
... Inhibitory activity of pepper leaves extracts(PLE) was determined according to Kim et al. [27] with modifications. Leave extract, substrate, enzyme, and acarbose solution were prepared in 0.1 M sodium phosphate buffer. ...
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Metabolomics and in vitro α-glucosidase inhibitory (AGI) activities of pepper leaves were used to identify bioactive compounds and select genotypes for the management of type 2 diabetes mellitus (T2DM). Targeted metabolite analysis using UPLC-DAD-QToF-MS was employed and identified compounds that belong to flavone and hydroxycinnamic acid derivatives from extracts of pepper leaves. A total of 21 metabolites were detected from 155 samples and identified based on MS fragmentations, retention time, UV absorbance, and previous reports. Apigenin-O-(malonyl) hexoside, luteolin-O-(malonyl) hexoside, and chrysoeriol-O-(malonyl) hexoside were identified for the first time from pepper leaves. Pepper genotypes showed a huge variation in their inhibitory activity against α-glucosidase enzyme(AGE) ranging from 17% to 79%. Genotype GP38 with inhib-itory activity of 79% was found to be more potent than the positive control acarbose (70.8%.). Orthogonal partial least square (OPLS) analyses were conducted for the prediction of the AGI activities of pepper leaves based on their metabolite composition. Compounds that contributed the most to the bioactivity prediction model (VIP >1.5), showed a strong inhibitory potency. Caffeoyl-putrescine was found to show a stronger inhibitory potency (IC50 = 145 µM) compared to acarbose (IC50 = 197 µM). The chemometric procedure combined with high-throughput AGI screening was effective in selecting polyphenols of pepper leaf for T2DM management.
... 28 Duckweeds can have high levels of avonoids and together with its nutritional value of amino acids and proteins make it suitable for human consumption with health benets. [29][30][31][32][33][34] Duckweeds have been sources of human food in several Asian countries. 35 Moreover, one duckweed species (Spirodela polyrhiza) is being used to treat urticaria, acute nephritis, inuenza, and inammation in Japan, Korea, and China. ...
... The dominant presence of C-glycosylated avonoids in duckweeds hypothetically protect these aquatic plants from oxidative stress and predators due to the lack of lignin and high metabolic ux towards phenylalanine and avonoids. 34 Lignin is a phenolic compound responsible for plant mechanical support and pathogen protection in plants. [56][57][58][59] Since duckweeds have trace amounts of lignin in all species 60-63 with the absence of vessels, other biomolecules such as avonoids are produced in higher quantities and possibly helps to protect duckweed tissues from pathogens. ...
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Duckweeds are the smallest free-floating flowering aquatic plants. Their biotechnological applications include their use as food, bioenergy, and environmental sustainability, as they can help clean polluted water. The high growth capacity and their chemical properties make them suitable for human health applications. Here we evaluated the ethanolic extracts from five species of duckweeds by HPLC-DAD/MS-MS for chemical characterization. Sixteen compounds were identified and quantified, in which three were chlorogenic acid derivatives and eleven apigenin and luteolin derivatives. We describe for the first time the presence in duckweeds of 5-O-(E)-caffeoylquinic acid (1), 3-O-(E)-coumaroylquinic acid (2), luteolin-7-O-glucoside-C-glucoside (3), 4-O-(E)-coumaroylquinic acid (4), luteolin-6-C-glucoside-8-C-rhamnoside (5), and luteolin-8-C-glucoside-6-C-rhamnoside (6). The flavonoids diversity showed a significant content of luteolin and its derivatives, except for Landoltia punctata that had significant apigenin content. Flavones identified in duckweeds were mostly C-glycosides, which can benefit human diets, and its abundance seems to be related to the higher antioxidant and anticancer capacities of Wolffiella caudata, Wolffia borealis, and Landoltia punctata. Our findings reinforce the idea that duckweeds could be valuable additives to the human diet, and their potential should be further explored.
... Unlike S. polyrhiza, which develops a seed-like turion for storing starch [33], L. punctata has a different storage mechanism. Metabolic pathway analysis showed that nutrient starvation and uniconazole treatment enhance starch anabolism but weaken starch catabolism in L. punctata, resulting in high starch accumulation by modulating the global carbon metabolism flux [34]. ...
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Full-text available
Background: Landoltia 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. Results: Comparative 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. Conclusions: This 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.
... Rhoifolin synthesis uses the formal reaction product (i.e., comosin) by flavanone-7-O-glucoside 2 -O-beta-l-rhamnosyltransferase (C12RT1) (Kumar et al., 2013;Lou et al., 2014). Prunin is also generated from naringenin by flavanone-7-O-beta-glucosyltransferase (GL) before naringin, which is considered the final metabolite (Tao et al., 2017). ...
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Bamboo, being an ornamental plant, has myriad aesthetic and economic significance. Particularly, Phyllostachys violascens cv. Viridisulcata contains an internode color phenotype in variation in green and yellow color between the sulcus and culm, respectively. This color variation is unique, but the underlying regulatory mechanism is still unknown. In this study, we used metabolomic and transcriptomic strategies to reveal the underlying mechanism of variation in internode color. A total of 81 metabolites were identified, and among those, prunin as a flavanone and rhoifolin as a flavone were discovered at a high level in the culm. We also found 424 differentially expressed genes and investigated three genes (PvGL, PvUF7GT, and PvC12RT1) that might be involved in prunin or rhoifolin biosynthesis. Their validation by qRT-PCR confirmed high transcript levels in the culm. The results revealed that PvGL, PvUF7GT, and PvC12RT1 might promote the accumulation of prunin and rhoifolin which were responsible for the variation in internode color of P. violascens. Our study also provides a glimpse into phenotypic coloration and is also a valuable resource for future studies.
... For example, in the subtropical climate of Eastern China, L. punctata is the first duckweed species to colonize water reservoirs in the Spring and the last remaining in the Fall, thus exhibiting the longest vegetative growth period compared with other duckweeds. Based on these qualities, the species has attracted much attention as a promising, inexpensive, and sustainable source of valuable biomass for the production of biofuels, such as ethanol, butanol, biogas, and hydrogen (Tao et al., 2017;Toyama et al., 2018;Miranda et al., 2020), and high-value biochemicals, such as succinic acid (Shen et al., 2018). ...
Article
Full-text available
Duckweeds are a group of monocotyledonous aquatic plants in the Araceae superfamily, represented by 37 species divided into five genera. Duckweeds are the fastest growing flowering plants and are distributed around the globe; moreover, these plants have multiple applications, including biomass production, wastewater remediation, and making pharmaceutical proteins. Dotted duckweed (Landoltia punctata), the sole species in genus Landoltia, is one of the most resilient duckweed species. The ribosomal DNA (rDNA) encodes the RNA components of ribosomes and represents a significant part of plant genomes but has not been comprehensively studied in duckweeds. Here, we characterized the 5S rDNA genes in L. punctata by cloning and sequencing 25 PCR fragments containing the 5S rDNA repeats. No length variation was detected in the 5S rDNA gene sequence, whereas the nontranscribed spacer (NTS) varied from 151 to 524 bp. The NTS variants were grouped into two major classes, which differed both in nucleotide sequence and the type and arrangement of the spacer subrepeats. The dominant class I NTS, with a characteristic 12-bp TC-rich sequence present in 3–18 copies, was classified into four subclasses, whereas the minor class II NTS, with shorter, 9-bp nucleotide repeats, was represented by two identical sequences. In addition to these diverse subrepeats, class I and class II NTSs differed in their representation of cis-elements and the patterns of predicted G-quadruplex structures, which may influence the transcription of the 5S rDNA. Similar to related duckweed species in the genus Spirodela, L. punctata has a relatively low rDNA copy number, but in contrast to Spirodela and the majority of other plants, the arrangement of the 5S rDNA units demonstrated an unusual, heterogeneous pattern in L. punctata, as revealed by analyzing clones containing double 5S rDNA neighboring units. Our findings may further stimulate the research on the evolution of the plant rDNA and discussion of the molecular forces driving homogenization of rDNA repeats in concerted evolution.
... 低氮胁迫能 够诱导少根紫萍(L. punctata)中参与淀粉和类黄酮生物 合成关键酶的基因表达上调, 而参与光合作用的酶和 木质化速率限制酶的表达却下调 [106,107] . [109] . ...
... An extensive list of the flavonoids identified in A. filiculoides and A. pinnata was previously shown [17,66]. Similarly to Azolla, the increase in the concentrations of flavonoid compounds was triggered in duckweed (Landoltia punctata) by nutrient starvation [67]. In terrestrial plants, the composition of flavonoids and their metabolic plasticity is mostly dependent on the plant species, their developmental stage, and environmental conditions, including both biotic and abiotic stresses [35]. ...
Article
Full-text available
The metabolic plasticity of shikimate and phenylpropanoid pathways redirects carbon flow to different sink products in order to protect sessile plants from environmental stresses. This study assessed the biochemical responses of two Azolla species, A. filiculoides and A. pinnata, to the combined effects of environmental and nutritional stresses experienced while growing outdoors under Australian summer conditions. These stresses triggered a more than 2-fold increase in the production of total phenols and their representatives, anthocyanins (up to 18-fold), flavonoids (up to 4.7-fold), and condensed tannins (up to 2.7-fold), which led to intense red coloration of the leaves. These changes were also associated with an increase in the concentration of carbohydrates and a decrease in concentrations of lipids and total proteins. Changes in lipid biosynthesis did not cause significant changes in concentrations of palmitoleic acid (C16:0), linolenic acid (C18:3), and linoleic acid (C18:2), the fatty acid signatures of Azolla species. However, a reduction in protein production triggered changes in biosynthesis of alanine, arginine, leucine, tyrosine, threonine, valine, and methionine amino acids. Stress-triggered changes in key nutritional components, phenolics, lipids, proteins, and carbohydrates could have a significant impact on the nutritional value of both Azolla species, which are widely used as a sustainable food supplement for livestock, poultry, and fish industries.
... With the rapid growth of global population, and the associated need for amino acids, duckweeds offer 20% to 45% of protein, consistent with WHO recommended amino acid intakes (Appenroth et al., 2017). Duckweeds are a source of high-quality animal protein substitutes, as well as several important vitamins (Kaplan et al., 2019), fat (4%-14%) (Yan et al., 2013), and starch (4%-75% of dry biomass) (Tao et al., 2017). In addition to their basic nutritional components, duckweeds have a high flavone glycoside content, predominantly consisting of apigenin-and luteolin-derived C-glycosides (Bai et al., 2018;Qiao et al., 2011;Wang, Xu et al., 2014), which are also present in certain vegetables and teas, such as rooibos tea (Krafczyk & Glomb, 2008), mung beans (Bai et al., 2016), legume leaves (Fu et al., 2008), and cucumber (Abou-Zaid, Lombardo, Kite, Grayer, & Veitch, 2001), and exhibit a broad range of bioactivities such as antioxidant, anti-inflammatory, anti-diabetic, neuroprotective, hepatoprotective, and antimicrobial (Xiao, Capanoglu, Jassbi, & Miron, 2016). ...
Article
Duckweeds have long been consumed as vegetables in several South Asian countries. In this study of the chemical constituents of duckweed Landoltia punctata, a new compound, apigenin 6-C-[β-D-apiofuranosyl-(1 → 2)]-β-D-glucopyranoside (1), and a previously LC-MS identified compound, quercetin 3-O-β-D-apiofuranoside (3), as well as three known compounds, luteolin 6-C-[β-D-apiofuranosyl-(1 → 2)]-β-D-glucopyranoside (2), apigenin 6-C-β-D-glucopyranoside (4), and luteolin 7-O-neohespirodise (5), were isolated and identified on the basis of MS and NMR spectroscopic analyses and chemical derivations. In total, 24 flavonoids were identified in L. punctata 0001 by UPLC-ESI-QTOF-MS2. In DPPH and ABTS assays, 3 exhibited significant antioxidant activity with IC50 values of 4.03 ± 1.31 µg/mL and 14.9 ± 2.28 µg/mL, respectively. In in vivo antioxidant activity assays, 1 significantly increased the survival rate of juglone-exposed Caenorhabditis elegans by 2 to 3-fold, and by 75% following thermal damage. Compounds 1-5 exhibited moderate scavenging capacities of intracellular reactive oxygen species in C. elegans exposed to H2O2.
Preprint
Full-text available
Duckweeds are fast-growing aquatic plants suitable for bioenergy due to fermentable-rich biomass with low lignin. The duckweed sub-families Lemnoideae and Wolffioideae are also distinguished by the distribution of two pectin classes (apiogalacturonan and xylogalacturonan), which seem to be related to their growing capacity and the starch content. The plant cell wall is built from pathways of nucleotide sugars syntheses that culminate in cell wall synthesis and deposition. Therefore, understanding these pathways through mapping the genes involved and their expression would be important to develop tools to improve bioenergy production. Here we used the available information of NDP-sugar metabolism to search for orthologous genes involved in the synthesis of cell wall polysaccharides in Spirodela polyrhiza . We detected 190 genes and mapped them onto the plant chromosomes . The genes were roughly arranged in groups according to their category: "Starch and sucrose metabolism," "Pectins," "Hemicelluloses," and "Cellulose." We followed the expression of thirty-eight of the orthologues’ transcripts – the higher expression being starch ( SBE ), pectin ( GAUT1, MUR, USP , and GER ), and mannan ( CSLA ) syntheses - corroborating the chemical composition of S. polyrhiza cell wall . We further investigated the carbohydrate metabolism pathways and discussed the implications of altering the NDP pathways for bioenergy and biorefinery. We conclude that S. polyrhiza displays suitable features for future genetic transformations leading to the adaptation of its cell wall for biofuels. However, such strategies will have to consider the trade-offs between fermentation and ethanol production benefits and the potential adverse effects of genetic transformation on plant growth and development.
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Ethnopharmacological relevance Dragon's Blood (Resina Draconis) is a red resin that has been used in traditional medicine to promote blood circulation, regenerate muscles, reduce swelling and pain, stop bleeding, etc., and its main chemical constituents are flavonoids. Dracaena cochinchinensis (Lour.) S.C.Chen is the only plant defined by the Pharmacopoeia of the People's Republic of China as a source of dragon's blood. Aim of the study We aimed to reveal genes involved in the biosynthesis and accumulation of flavonoids of D. cochinchinensis which is under wounding stress by performing a de novo transcriptome analysis. Materials and Methods D. cochinchinensis samples were collected for transcriptome sequencing and bioinformatics analysis at 0 days (0 d), 3 days (3 d), 6 days (6 d), and 10 days (10 d) after induction wounding stress, and tissues were microscopically observed after wounding stress. Results A total of 63,244 unigenes were obtained through bioinformatics analysis, and genes associated with the biosynthesis of flavonoids were identified. Through the analysis of DEGs after wounding stress in D. cochinchinensis, based on gene expression consistent with flavonoid accumulation levels, 20 genes in connection with the flavonoid synthesis pathway and 56 genes that may be responsible for flavonoid modification and transport, and also revealed TFs (MYB, bHLH) that may be responsible for flavonoid biosynthesis. Analysis of DEGs between the four periods revealed that after wounding stress, the greatest number of significant DEGs were enriched during the first 3 days, while fewer DEGs were enriched after day 3, which corresponding to only about 1/10 (353/3883) the number of DEGs during the first 3 days. In addition, putative unigenes involved in lignin biosynthesis, such as CSE, HCT, CCR, F5H, and CAD, were significantly down-regulation after D. cochinchinensis wounding stress, but the putative unigenes responsible for flavonoid biosynthesis, such as CHS, CHI, DFR, F3′5′H, F3H, ANR, FLS, and ANS were significantly up-regulation. Conclusion We performed de novo transcriptome analysis of D.cochinchinensis under wounding stress, candidate genes and TFs involved in the biosynthesis and accumulation of flavonoids were identified, which is the first report on the transcript variants in flavonoid form accumulation in D. cochinchinensis under wounding stress. According to the results of DEGs analysis, wounding stress attenuated lignin biosynthesis meanwhile promoted flavonoid biosynthesis. In addition, we also compared the transcriptomics of the two different original plants (D.cochinchinensis and D.cambodiana) that form dragon's blood in order to provide further understanding of the formation of dragon's blood.
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Summary: It is expected that emerging digital gene expression (DGE) technologies will overtake microarray technologies in the near future for many functional genomics applications. One of the fundamental data analysis tasks, especially for gene expression studies, involves determining whether there is evidence that counts for a transcript or exon are significantly different across experimental conditions. edgeR is a Bioconductor software package for examining differential expression of replicated count data. An overdispersed Poisson model is used to account for both biological and technical variability. Empirical Bayes methods are used to moderate the degree of overdispersion across transcripts, improving the reliability of inference. The methodology can be used even with the most minimal levels of replication, provided at least one phenotype or experimental condition is replicated. The software may have other applications beyond sequencing data, such as proteome peptide count data.Availability: The package is freely available under the LGPL licence from the Bioconductor web site (http://bioconductor.org).Contact: mrobinson@wehi.edu.au
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