14.2 Starch Production
and Proteomics
in Landoltia punctata
We previously determined Landoltia punctata
0202 was a candidate of highest biomass and
starch percentage strain under nutrient starvation
and uniconazole treatment through systematical
screening. When growing under the nutrient
starvation condition, high flavonoid and starch
accumulation can be achieved simultaneously in
L. punctata 0202. The flavonoids are a large
class of secondary metabolites widely distributed
in plants, which encompasses more than 10,000
structures, with different substituent groups,
including chalcones, flavones, flavonols, flavandiols, anthocyanins, condensed tannins, and
aurones (Winkel-Shirley 2001). The percentage
of total flavonoid increases up to 5.56% following nutrient starvation for 168 h, of which seven
components showed an obvious increase,
accompanied abundant anthocyanin with purple
coloration accumulated on the ventral side of
fronds of L. punctata 0202 (Huang et al. 2014;
Tao et al. 2017). Moreover, the cellulose and
lignin contents of duckweed are 9.25% and
3.84%, respectively, which were four times
lower than water hyacinth (39.93% and 10.15%,
respectively), indicating that duckweed has more
potential in animal food, fertilizer, and bioenergy
production than water hyacinth (Zhao et al.
2014).
Uniconazole is another factor contributing to
dry weight increase and high starch accumulation
of duckweed, the dry weight in one flask can
increase 3.1-fold compared to the control and the
starch content can increase up to 48% (15.2-fold
compared to the control) within 240 h after
spraying frond with 800 mg/L uniconazole, and
endogenous hormone content can be changed,
the contents of abscisic acid (ABA), cytokinin
(CK), and zeatin-riboside (ZR) increased, and on
the contrary, the content of gibberellin (GA) decreased with uniconazole application. Besides,
chlorophyll a and b content both increased
compared with the control, resulting in the photosynthetic rate elevated (Liu et al. 2015a, b;
Huang et al. 2015).
To elucidate the mechanisms of high starch
accumulation, quantitative proteomics was firstly
used to study the response of L. punctata 0202 to
nutrient starvation with iTRAQ-LC-MS/MS
technology. Duckweeds after expanding cultivation in sterile Hoagland nutrient solution for
14 days under stable condition were transferred
into distilled water for an additional 7 days in the
same condition, samples harvested on 11 time
points (0, 0.5, 2, 5, 24, 48, 72, 96, 120, 144, and
168 h) in time course were used for composition
characterization and enzymatic activity assay in
three biological replicates, and five time point
samples (0, 2, 5, 24, and 72 h) were subjected to
iTRAQ proteomic analysis. A total of 2015
unique proteins were identified based on the
duckweed protein sequence database using the
mRNA transcripts predicted by RNA-seq results.
In the identified proteins, 172 proteins were
up-regulated and 43 proteins were downregulated. Gene ontology (GO) categorization
analysis revealed that the biological process was
significantly enriched (76.7%) in the metabolic
process. Notably, in starch metabolism, the
expression levels of enzymes involved in starch
biosynthesis were up-regulated, whereas those
involved in starch degradation showed no significant difference. Importantly, in phenylpropanoid biosynthesis, the expression of several
key enzymes involved in flavonoid biosynthesis
showed up-regulated, but almost no enzyme
related to the lignin biosynthetic branch exhibited sufficient expression abundance for detection. The proteomic analysis directly and
powerfully demonstrated that high starch and
low lignin percentage were regulated by the
expression of enzymes and alteration of metabolic flux in the relevant pathways. This study
helps us to understand the molecular mechanism
of high starch accumulation and low lignin percentage in duckweed accurately, and promote the
development of duckweed as a bioenergy crop
(Huang et al. 2014).
Another proteomics research of duckweed is
to investigate uniconazole-induced phytohormone variation and starch accumulation in
L. punctata 0202. Duckweeds after expanding
cultivation in standard 1/6 Hoagland nutrient
14 Proteomics in Duckweeds
139
and Proteomics
in Landoltia punctata
We previously determined Landoltia punctata
0202 was a candidate of highest biomass and
starch percentage strain under nutrient starvation
and uniconazole treatment through systematical
screening. When growing under the nutrient
starvation condition, high flavonoid and starch
accumulation can be achieved simultaneously in
L. punctata 0202. The flavonoids are a large
class of secondary metabolites widely distributed
in plants, which encompasses more than 10,000
structures, with different substituent groups,
including chalcones, flavones, flavonols, flavandiols, anthocyanins, condensed tannins, and
aurones (Winkel-Shirley 2001). The percentage
of total flavonoid increases up to 5.56% following nutrient starvation for 168 h, of which seven
components showed an obvious increase,
accompanied abundant anthocyanin with purple
coloration accumulated on the ventral side of
fronds of L. punctata 0202 (Huang et al. 2014;
Tao et al. 2017). Moreover, the cellulose and
lignin contents of duckweed are 9.25% and
3.84%, respectively, which were four times
lower than water hyacinth (39.93% and 10.15%,
respectively), indicating that duckweed has more
potential in animal food, fertilizer, and bioenergy
production than water hyacinth (Zhao et al.
2014).
Uniconazole is another factor contributing to
dry weight increase and high starch accumulation
of duckweed, the dry weight in one flask can
increase 3.1-fold compared to the control and the
starch content can increase up to 48% (15.2-fold
compared to the control) within 240 h after
spraying frond with 800 mg/L uniconazole, and
endogenous hormone content can be changed,
the contents of abscisic acid (ABA), cytokinin
(CK), and zeatin-riboside (ZR) increased, and on
the contrary, the content of gibberellin (GA) decreased with uniconazole application. Besides,
chlorophyll a and b content both increased
compared with the control, resulting in the photosynthetic rate elevated (Liu et al. 2015a, b;
Huang et al. 2015).
To elucidate the mechanisms of high starch
accumulation, quantitative proteomics was firstly
used to study the response of L. punctata 0202 to
nutrient starvation with iTRAQ-LC-MS/MS
technology. Duckweeds after expanding cultivation in sterile Hoagland nutrient solution for
14 days under stable condition were transferred
into distilled water for an additional 7 days in the
same condition, samples harvested on 11 time
points (0, 0.5, 2, 5, 24, 48, 72, 96, 120, 144, and
168 h) in time course were used for composition
characterization and enzymatic activity assay in
three biological replicates, and five time point
samples (0, 2, 5, 24, and 72 h) were subjected to
iTRAQ proteomic analysis. A total of 2015
unique proteins were identified based on the
duckweed protein sequence database using the
mRNA transcripts predicted by RNA-seq results.
In the identified proteins, 172 proteins were
up-regulated and 43 proteins were downregulated. Gene ontology (GO) categorization
analysis revealed that the biological process was
significantly enriched (76.7%) in the metabolic
process. Notably, in starch metabolism, the
expression levels of enzymes involved in starch
biosynthesis were up-regulated, whereas those
involved in starch degradation showed no significant difference. Importantly, in phenylpropanoid biosynthesis, the expression of several
key enzymes involved in flavonoid biosynthesis
showed up-regulated, but almost no enzyme
related to the lignin biosynthetic branch exhibited sufficient expression abundance for detection. The proteomic analysis directly and
powerfully demonstrated that high starch and
low lignin percentage were regulated by the
expression of enzymes and alteration of metabolic flux in the relevant pathways. This study
helps us to understand the molecular mechanism
of high starch accumulation and low lignin percentage in duckweed accurately, and promote the
development of duckweed as a bioenergy crop
(Huang et al. 2014).
Another proteomics research of duckweed is
to investigate uniconazole-induced phytohormone variation and starch accumulation in
L. punctata 0202. Duckweeds after expanding
cultivation in standard 1/6 Hoagland nutrient
14 Proteomics in Duckweeds
139