Nitrate and Phosphate Regimes Induced Lipidomic and

Biochemical Changes in the Intertidal Macroalga

Ulva lactuca (Ulvophyceae, Chlorophyta)
1

Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002,
Gujarat, India
2
Institute of Plant Sciences, Agricultural Research Organization (ARO), The Volcani Center, PO Box 6, Bet Dagan 50250, Israel
*Corresponding author: E-mail, crk@csmcri.org; Fax, + 91-278-2567562/2566970.
(Received May 28, 2013; Accepted October 21, 2013)

This study was carried out in order to understand the lipid

and biochemical alterations resulting from different nutritional regimes of nitrate and phosphate in Ulva lactuca. The
algal thalli cultured in artificial seawater (ASW) showed
higher levels of carbohydrates and non-polar lipids and
increased phosphatase activities, accompanied by degradation of polar lipids, proteins and pigments. Further,
higher levels of lipid hydroperoxides indicated reative
oxygen species (ROS)-mediated non-enzymatic lipid peroxidation due to nutritional limitation-induced oxidative
stress. Those thalli cultured in ASW supplemented
with nitrate showed responses corresponding to nitrate
addition, such as an increase in pigments, monogalactosyldiacylglycerols, polyunsaturated fatty acids and nitrate
reductase. In addition, these thalli showed partial induction
of phosphatases, low phospholipids, and high sulfolipid
and 1,2-diacylglyceryl-3-O-40 -(N,N,N-trimethyl)-homoserine
(DGTS) due to phosphate limitation. Similarly, algal thalli
cultured in ASW supplemented with phosphate showed
down-regulation of phosphatases, an increase in phospholipids due to availability of phosphate as well as a decrease in
nitrate reductase, pigment, monogalactosyldiacylglycerols
and polyunsaturated fatty acids due to nitrate limitation.
On the other hand, algal thalli cultured in ASW supplemented with both nitrate and phosphate showed recovery
of lost pigments and proteins, a high monogalactosyldiacylglycerol/digalactosyldiacylglycerol ratio, high unsaturation
and high oxylipin levels (both C18 and C20). Further, the
accumulation of indole-3-acetic acid in nutrient-limited
thalli and of kinetin and kinetin riboside in nutrient-supplemented thalli indicated their antagonistic roles under
nutrient stress. Thus, U. lactuca copes with nitrate and
phosphate nutritional stress by altering the metabolic
pathways involved in lipid biosynthesis including a shift
in lipid classes, fatty acids, oxylipins and indole-3-acetic
acid/kinetin cross-talk.

Keywords: Lipids  Nitrate  Oxylipins  Phosphate  PUFAs

Ulva lactuca.

Abbreviations: AA, arachidonic acid; ALA, a-linolenic acid;

ALA-LOX, linolenate lipoxygenase; ALP, alkaline phosphatase;
AP, acid phosphatase; ASW, artificial seawater; DAG,
diacylglycerol; DGDG, digalactosyldiacylglycerol; DGTS, 1,
2-diacylglyceryl-3-O-40 -(N,N,N-trimethyl)-homoserine;
EPA,
eicosapentaenoic acid; ESI-MS, electrospray ionization-mass
spectrometry; EST, expressed sequence tag; FA, fatty acid;
GL, glycolipid; HETE, hydroxyoeicosatetraenoic acid; HODE,
hydroxyoctadecadienoic acid; HOTrE, hydroxyoctadecatrienoic acid; HpODE, hydroperoxyoctadecadienoic acid; IAA,
indole acetic acid; LA, linoleic acid; LA-LOX, linoleate lipoxygenase; LPC, lyso-phosphatidylcholine; LPE, lyso-phosphatidylethanolamine; LPG, lyso-phosphatidylglycerol; LOX,
lipoxygenase; MGDG, monogalactosyldiacylglycerol; MUFA,
monounsaturated fatty acid; NL, non-polar lipid; NR, nitrate
reductase; Pi, inorganic phosphate; PA, phosphatidic acid; PC,
phosphatidylcholine; PCA, principal component analysis; PE,
phosphatidylethanolamine; PG, phosphatidylglycerol; PGRs,
Plant growth regulators; PI, phosphatidylinositol; PL,
phospholipid; PS, phosphatidylserine; ROS, reactive oxygen
species; SFA, saturated fatty acid; SQDG, sulfoquinovosyldiacylglycerol; TAG, triacylglycerol; TFAs, total fatty acids;
THPOs, total hydroperoxy oxylipins; TLs, total lipids; Toxls,
total oxylipins.

Introduction
Macroalgae in the intertidal region often experience rapid
variations in temperature, salinity, light and nutrients (nitrate
and phosphate) that influence their physiology (Ale et al. 2011).
The differential nitrate and phosphate uptake and their storage are part of adaptive strategies evolved by macroalgae for
their successful sustenance. Changes in nitrate and phosphate

Plant Cell Physiol. 55(1): 5263 (2014) doi:10.1093/pcp/pct156, available online at www.pcp.oxfordjournals.org
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Regular Paper

Puja Kumari1, Manoj Kumar1,2, C.R.K. Reddy1,* and Bhavanath Jha1

Responses of Ulva lactuca under nutritional stress

PSI-driven electron transport flow, as well as the expression

of genes involved in carotene biosynthesis, antioxidant and
defense enzymes, nitric oxide signaling, Ca2+ release and early
light-induced gene expression in different Ulva spp. under
salinity, light and metal stress (Gonzalez et al. 2010, Gao et al.
2011, Dong et al. 2012, Gonzalez et al. 2012, Hsu and Lee 2012,
Xu et al. 2012, Zhang et al. 2012).
Ulva lactuca inhabits the intertidal region where the
availability of nitrate and phosphate in ocean varies dramatically on spatial and temporal scales, limiting the primary productivity over large areas. Several experiments have been
undertaken to study the uptake and assimilation of nitrate
and phosphate in the context of growth responses, pigments,
mineral composition and nutrient-assimilating enzymes in different species of Ulva (Lee and Tsai 2005, Robertson-Andersson
et al. 2009, Ale et al. 2011, Cabello-Pasini et al. 2011).
Nevertheless, a few studies have been undertaken on lipidomic
changes in Ulva spp. under nutritional stress, while the changes
in oxylipins, lipoxygenase (LOX) and phytohormones in the
context of nutrient stress have not been investigated.
Although earlier studies have largely dealt with physiological
responses to nitrate and phosphate stress individually, the
synergistic interaction of these two nutrients has remained
more or less unexplored. Therefore, an attempt was made in
this study to unravel the alterations in the lipidome, fatty
acids (FAs), oxylipins and LOX, as well as biochemical parameters including phytohormones, carbohydrates, protein and
nutrient assimilatory enzymes [nitrate reductase (NR) and
phosphatases] in U. lactuca cultured in different nutrients;
nitrate (N) and inorganic phosphate (P) individually and
together (NP). A particular effort was made to decipher the
discriminant responses of this alga that aid in its adaptation
to nutrient stress using multivariate analysis.

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concentrations cause coordinated adjustment of numerous

metabolic pathways, resulting in the reallocation of cellular
components such as starch, lipids and proteins (Hockin et al.
2012, Li et al. 2012). The depletion of these two nutrients also
initiates a cascade of physiological responses including a
decrease in photosynthetic and respiration rates, inhibition of
growth and activation of nutrient acquisition and the transport
network (Lee 2000, Lee and Tsai 2005, Young et al. 2009, Ale
et al. 2011). In addition, algal adaptation to nutrient changes
includes remodeling of their lipid composition (Guschina and
Harwood 2006). Nutritional constraint (carbon, nitrogen, phosphorus, sulfur and iron) has been demonstrated to be one
of the favorable strategies to enhance lipid production in
microalgae (Fan et al. 2012, Hockin et al. 2012, Li et al. 2012).
The decrease in phospholipid (PL) content accompanied by
an increase in non-phosphorus glycolipids (GLs) such as digalactosyldiacylglycerol (DGDG), sulfolipid, sulfoquinovosyldiacylglycerol (SQDG) and betaine lipids (especially in algae) is a
universal response to phosphate deprivation in algae and
higher plants (Benning et al. 1995, Hartel et al. 2000,
Andersson et al. 2003, Khozin-Goldberg and Cohen 2006,
Awai et al. 2007, Bellinger and Van Mooy 2012). Recently,
Okazaki et al. (2012) reported the role of a new class of plant
lipid, glucuronosyldiacylglycerols, in combating phosphate deprivation in Arabidopsis plants. Similarly, nitrate deprivation
leads to the accumulation of non-polar lipids (triacylglycerol;
TAG) while an increase in nitrate leads to an increase in galactolipid biosynthesis, especially monogalactosyldiacylglycerol
(MGDG) (Regnault et al. 1995, Guschina and Harwood 2006,
Fan et al. 2012, Li et al. 2012). In addition, Gaude et al. (2007)
demonstrated the accumulation of novel fatty acid phytyl
esters under nitrogen deficiency in Arabidopsis (Gaude et al.
2007). These lipid alterations have been reported to be regulated by auxin/cytokinin cross-talk in higher plants such as
Arabidopsis and maize under nitrate (Sakakibara, 2003) and
phosphate starvation (Kobayashi et al. 2006, Narise et al.
2010). Narise et al. (2010) showed that the expression of
genes involved in GL synthesis and PL degradation under inorganic phosphate (Pi) starvation was substantially reduced in
auxin signaling mutants, slr-1, a gain-of-function mutant of
IAA14 (a repressor of auxin signaling), and arf7arf19, a lossof-function mutant of auxin response factors ARF7 and
ARF19, further resulting in suppressed accumulation of
DGDG and SQDG. Lee et al. (2005) showed that phosphite
in Ulva lactuca interrupts the sensing mechanism during
phosphate-limiting conditions and inhibits the induction
of acid phosphatase (AP) isozymes and the high-affinity phosphate uptake system in phosphate-starved thalli. Nevertheless,
the hormonal regulation of lipid alterations under nitrate/
phosphate starvation is not known for macroalgae to date.
The genus Ulva has gained worldwide prominence recently
due to its high growth rate and innate ability to grow in wider
environmental conditions. It has emerged as one of the model
organisms for investigating complex metabolic networks such
as photosynthetic C3 and C4 pathways, ethylene production,

Results
Algal growth
The daily growth rate increased with the increase in concentration of each nutrient up to 20 mg l1 for nitrate and 10 mg l1
for phosphate, and thereafter it decreased gradually (Fig. 1).
Thus, the optimum concentrations of nitrate (20 mg l1) and
phosphate (10 mg l1) determined from the growth experiment
were applied in further studies. The daily growth rates of algal
thalli cultured with optimized nutrient concentrations (N, P
and NP) were 1.4- to 1.7-fold higher than those cultured only
in artificial seawater (ASW; P  0.05). This increase in growth
may be due to rapid uptake of nutrients by algae after 7 d of
nutrient starvation.

Proximate composition, pigment and

phytohormone analysis
There was a significant decrease in total carbohydrate
content (1.2- to 1.3-fold) with a concomitant increase in total
protein (1.3- to 1.6-fold) with the supplementation of nutrients

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P. Kumari et al.

Nitrate reductase, acid/alkaline phosphatase

enzyme activities

Fig. 1 Daily growth rate (DGR%) of Ulva lactuca cultured

under increasing nitrate (NaNO3) (A) and phosphate
(Na2HPO4H2O) concentration and (B) cultured with different
nutrient sources (C). DGR% values are reported as means of
triplicates (n = 3), and error bars show the SD among growth
rates of triplicate algal thalli cultured with each medium.
Different letters (a and b) indicate that the mean values
of the DGR for different nutrients were significantly different at
P  0.05.

The investigation of NR and phosphatase activities revealed

the synergistic effect of nitrate and phosphate in U. lactuca.
An increase in NR activity was observed in N- (1.87-fold) and
NP- (1.6-fold) supplemented thalli with the increase in nitrate
availability, while no significant change was observed in
ASW + P thalli (Fig. 2). On the other hand, there was a decrease
in both the AP and alkaline phosphatase (ALP) enzymes
with the availability of phosphate in P- (1.5- and 3.4-fold,
respectively) and NP- (1.7- and 3.8-fold, respectively) supplemented thalli.

Lipid and fatty acid analysis

The nutrient-supplemented algal thalli showed an increase
in total lipid (TL) content, with ASW + P thalli showing the

Table 1 Proximate composition, pigment and phytohormone contents of U. lactuca cultured with different nutrients
ASW

ASW + N

ASW + P

ASW + NP

C/N ratio

12.3 0.19a

10.2 0.18b,c

10.9 0.15b

Pi (% FW)

0.022 0.01b

0.025 0.003b

0.053 0.013a

0.048 0.01a

0.8 0.12b

0.82 0. 02b

1.82 0.04a

1.77 0.15a

Total P (% DW)
Carbohydrate (mg g1 FW)
Protein (mg g1 FW)

9.4 0.71c

60.78 3.0a

52.18 2.5b

48.3 1.7b

47.8 1.1c

6.2 0.8d

8.8 0.8c

8.0 0.1b

10.2 1.1a
372.4 3.5a

Pigments (mg g  1 FW)

Chl a

265.9 7.4b

276.7 2.3b

257.1 3.5b

Chl b

71.4 1.8b

101.7 2.6a

63.7 3.8b

108.0 9.1a

Carotenoids

83.4 1.3d

87.7 1.5c

90.5 2.9b

108.9 3.0a

3.7 0.1

2.7 0.1

4.2 1.2

3.5 0.3
177.7 24.0a

Chl a/b
Phytohormones

219.5 94.8a

191.2 73a

148.7 41.6a

Kinetin

0.36 0.03c

0.47 0.1b

0.38 0.1b,c

0.87 0.1

Kinetin riboside

12.4 1.1c

25.1 7.4a,b

18.8 3.2b,c

27.2 4.2a

Gibberellic acid

43.3 3.5b

68.1 15.7b

57.2 12.2b

81.8 16.1a

ABA

79.7 11.4a

54.0 9.3b

45.6 8.7b

53.5 5.7b

IAA

Values are expressed as the means SD (n = 3).

ac, values in the same row are significantly different at P  0.05.

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(Table 1). The nutrient-supplemented thalli showed an

increase in Chl a and Chl b, with the exception of ASW + P
thalli that showed a decrease in both Chl a and Chl b contents
(1.03- to 1.12-fold) (Table 1). However, the carotenoids
increased 1.1- to 1.3-fold with the supplementation of both
the nutrients. Phytohormone analysis showed the accumulation of auxin [indole acetic acid (IAA); 1.1- to 1.5-fold] and
ABA (1.5- to 1.7-fold) in nutrient-starved ASW-cultured thalli,
with a concomitant decrease in kinetin (1.05- to 2.4-fold),
kinetin riboside (1.5- to 2.2-fold) and gibberellic acid (1.3- to
1.9-fold). The decrease in IAA and ABA was in the order
ASW > ASW + N >ASW + NP > ASW + P, while the increase
in kinetin, kinetin riboside and gibberellic acid was in the
order ASW + NP > ASW + N > ASW + P > ASW (Table 1).

Responses of Ulva lactuca under nutritional stress

maximum increase of 1.6-fold (Table 2). The detailed analysis

of different lipid classes revealed the accumulation of nonpolar lipids (NLs; 1.3- to 1.5-fold) in ASW thalli as compared
with nutrient-supplemented thalli. Further, ASW + N and
ASW + NP thalli showed an increase of 1.5- and 1.1-fold in
GLs, respectively, as compared with ASW thalli, while
ASW + P thalli showed a 1.2-fold decrease in GLs due to N
limitation (Fig. 3). Similarly, ASW + P and ASW + NP
thalli showed a 1.4- and 1.2-fold increase, respectively, in PLs
in comparison with ASW-cultured thalli, while PL content
further decreased in ASW + N thalli due to prolonged Pi
limitation.
Furthermore, electrospray ionization-mass spectrometry
(ESI-MS) analysis of GLs revealed increased ion intensities of
DGDG (2.3- to 4.9-fold) in ASW thalli accompanied by SQDG
and DGTS accumulation (Fig. 3). However, ASW + N thalli
showed the maximum ion intensities for SQDG and DGTS
(1.9- and 1.2-fold, respectively), which indicated an adaptation
towards Pi limitation as their contents decreased in ASW + P
and ASW + NP thalli. MGDG showed a 1.4- to 2.4-fold increase
in all the nutrient-supplemented thalli in the order
ASW + N > ASW + NP > ASW + P > ASW (Fig. 3). The increase in the signals of MGDG 34:5, 34:6, 36:8 and 38:8 concomitant with the decrease in DGDG 34:5, 34:6, 36:8 and 38:8 in
nutrient-supplemented thalli pointed towards a decrease in the
conversion rate of MGDG-containing polyunsaturated fatty
acids (PUFAs) to those containing DGDG (Fig. 3). The ESI-MS
analysis of PLs showed increased ion intensities of PLs in the
order of phosphatidylglycerol (PG) > phosphatidylserine
(PS) > phosphatidylinositol (PI) > phosphatidylethamolamine
(PE) > phosphatidic acid (PA) in ASW + P and ASW + NP
thalli. In contrast, starved ASW thalli showed a 1.1- to 1.4fold increase in PA and lyso-products (LPG and LPE), indicating
lipid degradation (Fig. 3). Both ASW and ASW + N thalli
showed a decrease in PE (1.9- to 2.8-fold), PG (1.5- to 3.7fold), PS (3.0- to 4.2-fold) and PI (1.5- to 3.7-fold) due to Pi
limitation.

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Fig. 2 Effect of different nutrients on nitrate reductase (NR), acid

phosphatase (AP) and alkaline phosphatase (ALP) activities of Ulva
lactuca. The values are represented as means of triplicate data (n = 3),
and error bars show the SD. Different letters (ac) on columns with
the same shading indicate that mean values of enzyme activities for U.
lactuca cultured with different nutrients were significantly different at
P  0.05.

FA analysis revealed a 1.2- to 1.5-fold increase in PUFAs at

the expense of saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) in nutrient-supplemented thalli,
except in ASW + P thalli that showed a 1.1-fold increase in
SFAs mainly due to an increase in palmitic acid (C16:0)
(Table 2). This increase in PUFAs also shifted the PUFA/SFA
ratio from 0.71 : 1 in ASW thalli to 0.76 : 1 in ASW + P, 1.1 : 1 in
ASW + N and 1.58 : 1 in ASW + NP thalli. This increase in PUFAs
was mainly due to an increase in a-linolenic acid (ALA; C18:3,
n3) that contributed to 22.6% of the total fatty acids (TFAs)
in ASW + P (1.5-fold increase) to 28.3% of TFAs in ASW + N
(1.86-fold increase) and 32% of TFAs in ASW + NP thalli (2.1fold increase). The increase in ALA may be due to increased
availability of its substrates C18:2, n-6 [linoleic acid (LA)] that
may result in up-regulation of
15-desaturases responsible
for the conversion of LA to ALA. Moreover, the content of
C20 PUFAs increased in ASW + P (1.9-fold) and ASW + NP
(2.1-fold) thalli, mainly due to a 2.2- to 2.5-fold increase in
arachidonic acid (AA; C20:4, n6) and a 1.3- to 1.9-fold increase
in eicosapentaenoic acid (EPA; C20:5, n3) as compared with
ASW thalli.

Oxylipin content and lipoxygenase activity

The total oxylipin (Toxl) content increased 1.6- to 2.3-fold
(Fig. 4) with the addition of nutrients to the starved ASW
thalli, mainly due to the increase in the contents of hydroxyoctadecadienoic (HODEs) and hydroxyoctadecatrienoic acids
(HOTrEs). This increase in HODEs and HOTrEs may be due
to an increase in the availability of their substrates (C18
PUFAs) in nutrient-supplemented thalli. This increase in
HODEs, HOTrEs and hydroxyoeicosatetraenoic acids (HETEs)
was in agreement with the increased LOX activities obtained
with the three substrates LA (1.9- to 2.8-fold), ALA (4.6- to 8.4fold) and AA (2.8- to 4.2-fold) after the addition of nutrients
(Fig. 4). In contrast, the content of hydroperoxy oxylipins
decreased (1.03- to 1.6-fold) after nutrient supplementation,
indicating nutrient stress in ASW-cultured thalli. This indicated
the non-enzymatic peroxidation of FAs mediated by reactive
oxygen species (ROS) in ASW thalli that led to the accumulation of hydroperoxy oxylipins despite low LOX activity. Further,
the relative contents of 9- and 13-HODEs and HOTrEs showed
the up-regulation of linoleate 9-LOX and linolenate 13-LOX
on nutrient supplementation, that transform LA and ALA,
respectively, to their oxidized derivatives (Fig. 4). Similarly,
the relative contents of different HETEs indicated the upregulation of arachidonate 5-LOX in all the nutrientsupplemented thalli, and of arachidonate 11-LOX in
ASW + N- and arachidonate 8-, 12- and 15-LOX in ASW +
P-supplemented thalli (Supplementary Table S1).

Principal component analysis (PCA)

PCA revealed the global changes in the studied metabolome
of U. lactuca under different nutritional regimes. It explained
86% of the variations inherent in the data matrix (PC1, 54%
and PC2, 32%) (Fig. 5). ASW thalli were positively correlated

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P. Kumari et al.

Table 2 Lipid content and fatty acid composition of Ulva lactuca cultured with different nutrients
ASW
Lipid (mg g

FW)

13.2 1.1b,c

ASW + N

ASW + P

ASW + NP

15.2 4.5b

23.6 4.8a

14.0 2.0b,c

0.98 0.6a,b

0.11 0.03c

0.33 0.2b

5.5 1.3b,c

8.4 2.7b

4.16 0.4c

0.35 0.04

0.44 0.2

41.6 6.3

26.2 8

Fatty acids (% of total fatty acid methyl esters)

C12:0

1.53 0.07a
11.85 1.2a

C15:0

0.45 0.14

C16:0

33.5 2.2

C17:0

0.35 0.02

0.5 0.3

0.44 0.4

0.22 0.06

C18:0

3.5 0.6

6.8 0.6

4.7 0.8

3.5 0.1

C20:0

0.38 0.02

0.9 0.5

0.3 0.1

0.35 0.08

C22:0

1.4 0.1

0.52 0.3

1.3 0.5

1.3 0.32

C24:0

0.26 0.02

0.2 0.1

0.3 0.1

0.4 0.09

C16:1 (n7)

2.4 0.5

1.6 0.6

1.8 0.5

1.8 0.7

0.4 0.4
31.1 7

C17:1 (n7)

0.68 0.13a

0.2 0.15b

0.41 0.06b

C18:1 (n9)

4.5 0.3

4.2 1.4

2.85 1

C18:2 (n6)

11.8 0.24

12.3 2.9

C18:3 (n6)

0.26 0.1

0.4 0.07

C18:3 (n3)

15.2 1c

28.3 4.1a,b

0.5 0.1a,b
3.3 0.3

8.9 1.3

10.8 2.8

0.32 0.06

0.48 0.2

22.6 2.7b,c

32 7.1a

C18:4 (n-)

6.2 1.5

6.5 1.4

7.3 2.6

7.8 1.8

C20:3: (n6)

0.4 0.03a,b

0.3 0.2b

0.25 0.06b

0.7 0.3a

C20:4 (n6)

0.5 0.1b

0.4 0.3b

1.3 0.6a

0.95 0.4a,b

C20:5 (n3)

0.53 0.02b

0.44 0.3b

1.1 0.2a

0.67 0.2b

C22:6 (n3)

2.2 0.5

1.2 0.7

1.8 0.6

1.9 0.4

C22:4 (n6)

0.11 0.01b

0.04 0.02c

0.14 0.02a

0.02 0.01c

C18:2 (n6)/C18:1 (n9)

2.66 0.8

3.2 1.3

3.1 1.2

3.2 0.8

C18:3 (n6)/C18:2 (n6)

0.02 0.01

0.03 0.01

0.04 0.01

0.04 0.01

C18:3 (n3)/C18:2 (n6)

1.28 0.08b

C18:4 (n3)/C18:3 (n3)

0.4 0.1a

0.23 0.06b

C20:3 (n6)/C18:3 (n6)

1.5 0.5

0.8 0.7

0.8 0.1

1.5 0.7

C20:4 (n6)/C20:3 (n6)

1.5 0.3b

1.3 0.1b

5.1 2.2a

1.4 0.2b

2.3 0.22a,b

3.05 + 0.7a

0.3 0.07a,b

0.24 0.05b

SFA

53.2 2.4

MUFA

10.7 0.9a

PUFA

37.9 1.9

50.04 6.5

44.1 4.8

55.2 8.1

0.7 0.1

0.95 0.5

0.8 0.3

1.5 0.7

PUFA/SFA

46.9 8.6

2.6 0.3a,b

8.2 2.4a,b

58.6 3.7
5.2 2.2b

36.9 8.5
7.5 0.6a,b

Values are expressed as the means SD (n = 3).

ac, values in the same row are significantly different at P  0.05.

with NLs, DGDG, PA, LPE, SFAs, MUFAs, total hydroperoxy

lipins (THPOs), total carbohydrate content, AP, ALP, and
the phytohormones IAA and ABA. This validated our findings
that U. lactuca adapts to nutritional deprivation of nitrate and
phosphate by synthesizing more carbohydrates, that are further
converted to non-polar lipids (TAG), accumulation of hydroperoxides, a decrease in unsaturation, increased synthesis of
DGDG as well as degradation of PLs to PA/lysolipids. In addition, there was an up-regulation of phosphatases to make
phosphate available for thalli. ASW + N thalli were positively
correlated with total GLs, MGDG, SQDG, DGTS, LPG, NR and
Chl b, further confirming that the algal thalli show increased
synthesis of NR and GLs, especially MGDG, on availability of
nitrate, at the same time also showing the symptoms of

56

phosphate deficiency (accumulation of DGDG and SQDG).

ASW + P thalli were positively correlated with total PLs, PG,
PI, PE, PS, C20 PUFAs and total HETEs, indicating that with
the availability of phosphate, PL synthesis is remarkably
increased in U. lactuca along with the production of C20
PUFAs and their derivatives HETEs. In contrast, ASW + NP
thalli were positively correlated with PUFAs (mainly C18
PUFAs), total HODEs, total HOTrEs, LOX, Chl a, carotenoids,
protein and the phytohormones kinetin, kinetin riboside and
gibberellic acid due to their higher loadings and these metabolites showed the most remarkable changes when both the
nutrients were available. These positive correlations of treated
U. lactuca thalli with different variables signify their adaptations
towards available nutrients.

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C14:0

Responses of Ulva lactuca under nutritional stress

Discussion
Ulva lactuca responded to different nutritional regimes of
nitrate and phosphate by undergoing a shift in the proportion
of carbohydrate, lipids and proteins, together with metabolic
alterations in pigments, phytohormones, FAs and oxylipins.

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Fig. 3 Effect of different nutrients on the lipidome of Ulva lactuca.

(A) Changes in relative contents of different lipid classes, NL
(non-polar lipids), GL (glycolipids) and PL (phospholipids); (B) glycolipids: MGDG (monogalactosyldiacyl glycerol), DGDG (digalactosyldiacylglycerol), SQDG (sulfoquinovosyldiacylglycerol), betaine lipid
DGTS [1, 2-diacylglyceryl-3-O-40 -(N,N,N-trimethyl)-homoserine]; and
(C) phospholipids: PC (phosphatidylcholine), PE (phosphatidylethanolamine), PG (phosphatidylglycerol), PS (phosphatidylserine), PI
(phosphatidylinositol), PA (phosphatidic acid), LPC (lyso- phosphatidylcholine), LPG (lyso-phosphatidylglycerol) and LPE (lyso-phosphatidylethanolamine). Phosphatidylcholine (PC) was not detected.
The values are represented as means of triplicate data data (n = 3),
and error bars show the SD. Different letters (ac) on columns with
the same shading indicate that mean values for different nutrients
were significantly different at P  0.05.

The increase in the daily growth after nutrient supplementation

in U. lactuca (Fig. 1) could be attributed to the rapid uptake of
nutrients by starved thalli (Kamer et al. 2004, Young et al. 2009).
This was in agreement with the tissue N and P levels, with the
highest content of tissue N obtained in ASW + NP thalli regardless of Pi addition, similar to the report of Kamer et al. (2004) in
U. intestinalis, while the highest tissue P content (Table 1) was
observed in ASW + P (Lee 2000). The slight decrease in tissue P
content in ASW + NP as compared with ASW + P thalli may be
due to the partial inhibition of phosphate uptake by nitrate by
binding to or blocking the phosphate transporter (Lundberg
et al. 1989). This resulted in the maximum growth of ASW + P
thalli, followed by ASW + NP and ASW + N, indicating a state of
Pi limitation in U. lactuca once the N limitation is relieved
(Kamer et al. 2004, Teichberg et al. 2010). Villares et al. (1999)
also previously found a greater correlation between tissue P
(r = 0.68) and growth as compared with N (r = 0.61), illustrating
that P plays a more important role in the regulation of growth
than other nutrients and thus could be a limiting nutrient for
the primary production of Ulva sp.
There was a decrease in total carbohydrate content
concomitant with an increase in total protein and photosynthetic pigments (Table 1), in agreement with the earlier reports
in macroalgae (Gomez Pinchetti et al. 1998, Young et al. 2009,
Cabello-Pasini et al. 2011). However, ASW + P thalli showed a
decrease in the contents of Chl a and Chl b due to N depletion
as algal thalli were cultured in ASW for 7 d and then supplemented only with Pi, and this would have led to the state of
N limitation in ASW + P thalli. Latasa and Berdalet (1994)
also showed greater loss of photosynthetic pigments under
N deficiency in comparison with Pi deficiency in Heterocapsa
sp., signifying the strong relationship between pigment synthesis and N metabolism. Recently, Hockin et al. (2012) detected a
1.5- to 2.5-fold decrease in five proteins involved in the synthesis of Chl and its precursors in the proteome of the N-starved
diatom Thalassiosira pseudonana.
The increase in NR activity in ASW + N and ASW + NP thalli
resulting from the opportunistic uptake of nitrate with the
availability of nitrate (Fig. 2) was in accordance with the earlier
observations reported in Ulva sp., Gracilaria chilensis and
Fucus sp. (Chow and de Oliveira 2008, Young et al. 2009). The
decrease in AP and ALP activities with the increase in Pi and
total P contents in phosphate-supplemented thalli signified
the induction of phosphatase activities in starved ASW and
ASW + N thalli to degrade cellular stored P compounds or
medium soluble organic P compounds to compensate the P
requirement as a notable consequence of P limitation (Lee
2000, Lee and Tsai 2005). Moreover, the AP activity was > 10
times higher than the ALP activity, indicating that AP is first
induced to liberate Pi from intracellular P reserves and extracellular ALP is induced only when the intracellular Pi pool is
exhausted to utilize dissolved organic P compounds from the
medium (Lee 2000).
The alteration in lipid content is one of the important
adaptation strategies to nutrient imbalance in algae. In the

57

P. Kumari et al.

Fig. 5 Principal component analysis depicting discriminant responses of Ulva lactuca cultured with different nutrients with the first two
principal components.

present study, the highest lipid content was shown by Nstarved ASW + P thalli followed by Pi-starved ASW + N thalli,
indicating higher lipid accumulation under nutrient starvation
(Floreto et al. 1996, Guschina and Harwood 2006, KhozinGoldberg and Cohen 2006, Dean et al. 2010, Cakmak et al.
rdog et al. 2012). However, ASW
2012, Feng et al. 2012, O
thalli had the highest carbohydrate content instead of lipids,
indicating that up-regulation of carbohydrate synthesis is the
first response to nutrient deprivation and it seems from the
information available in the literature that carbon skeletons are
channeled to FA synthesis in the later stage of starvation where

58

carbohydrates are degraded to support FA biosynthesis

(Rismani-Yazdi et al. 2011, Cakmak et al. 2012, Recht et al.
2012). Recently, Fan et al. (2012) showed that lipid synthesis
lags behind that of starch in N-starved Chlamydomonas reinhardtii and emphasized that lipid synthesis occurs only when
the carbon supply exceeds the capacity for starch synthesis.
Thus, it seems that 7 d of nutrient starvation may not be sufficient to shift carbon metabolism towards FAs in ASW thalli in
this study. When U. lactuca thalli starved for 7 d were supplemented with sustained addition of either N or Pi, they showed
an increase in growth due to rapid uptake of available N/Pi but,

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Fig. 4 Effect of different nutrients on oxylipin contents and lipoxygenase (LOX) activity in Ulva lactuca. (A) Hydroxyoctadecadienoic acids
(HODEs); (B) hydroxyoctadecatrienoic acids (HOTrEs); (C) hydroperoxyoctadecadienoic acids (HpODEs); (D) oxylipin groups: THODEs (total
hydroxyoctadecadienoic acids), THOTrEs (total hydroxyoctadecatrienoic acids), THETEs (total hydroxyoeicosatetraenoic acids) and THPOs
(total hydroperoxy oxylipins); and (E) LOX activity with different substrates, linoleic acid (LA), a-linolenic acid (ALA) and arachidonic acid
(AA). The values are represented as means of triplicate data (n = 3), and error bars show the SD. Different letters (ac) on columns with the same
shading indicate that mean values for different nutrients were significantly different at P  0.05.

Responses of Ulva lactuca under nutritional stress

under Pi starvation (Hartel et al. 2000, Andersson et al. 2003,

Nakamura et al. 2005, Khozin-Goldberg and Cohen 2006, Awai
et al. 2007, Van Mooy et al. 2009, Narise et al. 2010). This lipid
remodeling under Pi starvation in higher plants has been documented to be mediated by the up-regulation of phospholipases
(PLD a1, a2, z1 and z2) and non-specific phospholipase C
(NPC4 and 5) that hydrolyze PLs to PA and diacylglycerol
(DAG). DAG is further utilized to synthesize GLs by MGDG
synthases (MGD2 and 3) that are expressed only under stress,
DGDG synthases (DGD 1 and 2) and SGDG synthases (SQD1
and 2) (Yu et al. 2002, Nakamura et al. 2005, Kobayashi et al.
2006, Gaude et al. 2008). In this study, PA ranged from 5% to 9%
of total PL ion intensities among nutrient-supplemented thalli,
in agreement with 410% of PL contents in earlier studies
(Dembitsky and Rozentsvet 1989) and our personal findings
(unpublished results). PA content increased up to 15% of
total PL ion intensities in nutrient-deprived ASW thalli due to
PL degradation under nutrient deprivation. Miller et al. (2010)
also found increased transcript levels of genes for PA in expressed sequence tags (ESTs) of nitrogen-deplete C. reinhardtii.
However, the low PA content shown by ASW + N thalli despite
the highest lyso-phospholipid content may be attributed to
the PA degradation by PA phosphatase to maintain a low PL
content due to prolonged Pi starvation and simultaneous
utilization of the glycerol backbone for GL synthesis
(Andersson et al. 2005) as evident from the increase in
GL content in ASW + N thalli. Similarly, higher plants
and algae cope with N starvation by synthesis of TAG
and higher DGDG/MGDG (Regnault et al. 1995, Gaude et al.
2007). In contrast, ASW + P thalli showed an increased
synthesis of PLs due to availability of Pi, but the MGDG
content was found to be reduced (1.3- to 1.8-fold) in comparison with N-supplemented ASW + N and ASW + NP thalli
(Fig. 3).
The flux in membrane lipid composition in higher plants is
regulated by the cross-talk between auxin and cytokinin
(Kobayashi et al. 2006, Narise et al. 2010). Kobayashi et al.
(2006) reported that treatment with auxin transport inhibitors
or the mutation of auxin transport genes decreased MGD2/3
transcription in Arabidopsis during Pi starvation and inhibited
the accumulation of DGDG. On the other hand, cytokinin
repressed the expression of MGD2/3 genes during Pi starvation,
acting antagonistically to auxin. The increased content of IAA
in ASW and ASW + N thalli in comparison with ASW + P and
ASW + NP thalli indicates a similar role for IAA in algae analogous to that in the higher plants. The increased contents of
kinetin and kinetin riboside with the addition of nutrients were
in agreement with the reports of Sakakibara (2003) and Rahayu
et al. (2005) in higher plants. As there is no study deciphering
the role of plant growth regulators (PGRs) under nutritional
stress in algae, it would be very premature to hypothesize their
significance in adaptation to nutritional stress merely on
the basis of their relative contents, but mutational studies in
combination with transcriptional analysis would certainly
prove their role.

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at the same time, they were subjected to the nutrient limitation

for the other nutrient, Pi limitation in ASW + N thalli and N
limitation in ASW + P thalli, resulting in utilization of the carbohydrate pool for lipid biosynthesis. Similar findings were
obtained by Feng et al. (2012) in Chlorella zofingngiensis wherein
they reported that NP+ cultures showed the highest lipid
content of 65% dry cell weight (dcw), N+P cultures had
44.7% lipid dcw, while N+P+ cultures had 33.5% dcw lipid
content. ASW + N showed higher contents of GLs concomitant
with low content of PLs due to P limitation in order to conserve
P by reducing the amount of P bound to membrane lipids
without compromising the function of photosynthetic membranes, while ASW + P showed higher contents of PLs, in agreement with the earlier reports in algae and higher plants
(Regnault et al. 1995, Hartel et al. 2000, Andersson et al. 2003,
Guschina and Harwood 2006, Khozin-Goldberg and Cohen
2006, Fan et al. 2012, Li et al. 2012). ASW + NP showed the
mobilization of FAs from NL to both the GLs and PLs due to
availability of the nutrients (Fig. 3). Furthermore, the GLs were
not the dominant fraction in U. lactuca, even those cultured
with both the nutrients (nitrate and phosphate); instead PLs
represented 41.8% of the TLs followed by GLs (30.45% of TLs).
Such a low GL content was in agreement with the earlier reports of low GL contents of 10.930% of TLs in macroalgae,
including as low as 10.9% of TLs for Ulva sp. (Dembitsky et al.
1990, Hossain et al. 2003, Shevchenko et al. 2007, El-Baroty et al.
2011). However, another group of researchers have reported
GLs to be the major fraction in macroalgae (Khotimchenko
2002, Khotimchenko 2003, Sanina et al. 2004), indicating
that there is a large variation in GL values reported in the
literature (as low as 10% to as high as >50% of TLs). We have
estimated the lipid class composition of different green, red
and brown algae in a separate experiment, where we found
that GLs ranged from 16% to 34% of TLs among Ulva spp.,
13.638.4% among Gracilaria spp. and 25.936.8% among
different brown macroalgal species (unpublished results).
Moreover, the lipid class composition varies with algal species,
their nutritional status, developmental stages and also
environmental factors, and thus a low GL content is not
unusual for Ulva spp. reported here.
Further, ESI-MS analysis of polar lipids enabled us to monitor
the lipidomic alterations at the level of individual lipid classes. A
decrease in the ratio of MGDG/DGDG glycolipid ion intensities
(5.6- to 9.8-fold) was observed in ASW thalli to stabilize the
physical state of the chloroplast membrane and the protein
pigment complex of the photosynthetic apparatus as MGDG is
non-bilayer forming, in contrast to DGDG (Regnault et al. 1995,
Dormann and Benning 2002). In addition to the increase in
DGDG glycolipid ion intensity in ASW (2.3- to 4.9-fold) and
ASW + N (1.02- to 1.1-fold) thalli, an increase in SQDG glycolipid ion intensity (1.1- to 2.1-fold) and DGTS ion intensity (1.8to 2.8-fold) accompanied by a decrease in PG phospholipid ion
intensity (2.2- to 5.5-fold) and PE phospholipid ion intensity
(1.9- to 5.6-fold) (Fig. 3) was observed, and these are considered
as one of the adaptation strategies of higher plants and algae

59

P. Kumari et al.

60

11-LOX in ASW + N and arachidonate 8-, 12- and 15-LOX in

ASW + P thalli.
PCA is a mathematical algorithm that reduces the dimensionality of the data while retaining most of the variation in the
data set. The data are represented by principal components,
with the first principal component accounting for the
maximum possible variation in the data, and each succeeding
component accounting for a portion of the remaining possible
variation. The plots allow visual assessment of the similarities
and differences among samples and help to determine whether
and how samples can be grouped. PCA was employed in
the present study to obtain a comprehensive view of algal
responses to nitrate and phosphate limitation that enabled
them to adapt to nutritional stress as well as to validate
our findings. It revealed that the deficiency of both N and Pi
leads to the accumulation of NLs and carbohydrates, an
increase in the DGDG/MGDG ratio concomitant with the degradation of PLs, leading to the increased concentration of PA
and lyso-products. In addition, there is a down-regulation of
desaturases resulting in accumulation of SFA and MUFA,
degradation of pigments, down-regulation of NR as well as
co-activation of phosphatase enzymes and an increase in lipid
hydroperoxides due to nutritional imbalance-induced oxidative
stress. On the other hand, the supplementation of nutrients
resulted in retrieval of pigments, PUFA, MGDG and PL contents
along with up-regulation of NR and down-regulation of
phosphatases. This cascade of metabolic responses was also
accompanied by phytohormone responses, where the effects
of IAA and kinetin were found to be antagonistic to each other,
with IAA accumulation in nutrient starvation and increased
kinetin contents after the addition of nutrients, analogous to
higher plants. We would like to mention here that these physiological changes in U. lactuca in response to nutrient stress of
nitrate and phosphate were observed in the algal thalli cultured
in the laboratory under controlled conditions. It is presumed
that similar metabolic alterations may take place in algae in
the field when there are fluctuations in nutrients (nitrate and
phosphate), as observed in laboratory conditions. However,
mesocosm studies in the field could show a more realistic picture of metabolic alterations in U. lactuca under nutritional
fluctuations in an intertidal region.
Mission et al. (2005) have also previously deciphered induction of genes for Pi transporters, AP genes, Pi salvaging from
organic compounds, PL degradation, GL synthesis, phytohormone responses, protein degradation as well as suppression
of genes involved in lipid synthesis, reactive oxygen control
and protein synthesis. Recently, Li et al. (2012) found 174
N deficiency-induced ESTs involved in carbohydrate metabolism, pyruvate and acetyl-CoA synthesis, isoprenoid degradation
and TAG accumulation, as well as down-regulation in the
expression of 116 genes related to photosynthesis, cell
growth, amino acid synthesis and cell cycle regulation in the
green alga Micractinium pusillum.
In conclusion, the present study revealed that U. lactuca
cope with nitrate and phosphate nutritional stress by altering

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FA analysis revealed a typical profile of Ulvales members

with higher unsaturation; C18 > C20 PUFAs (Kumari et al.
2013a) in all the nutritional conditions. There was an increase
in PUFAs (mainly ALA mediated by the up-regulation of
15
desaturases) in N-supplemented but Pi-limited ASW + N thalli
and ASW + NP thalli accompanied by a decrease in SFAs and
MUFAs. In contrast, N-limited and Pi-supplemented ASW + P
showed a 1.1- to 1.6-fold increase in SFAs, mainly due to an
increase in palmitic acid (1.2- to 1.6-fold) with a concomitant
decrease in unsaturated FAs. A similar increase in SFA accompanied by a decrease in unsaturated FAs under N starvation
and an increase in PUFAs at the expense of SFAs and MUFAs
under P starvation has been demonstrated in U. pertusa
(Floreto et al. 1995), Euglena gracilis (Regnault et al. 1995),
Chlorella spp. (Elsheek and Rady 1995, Liang et al. 2012),
Tetraselmis subcordiformis, Nannochloropsis occulata, Pavlova
viridis (Dean et al. 2010) and Dunaliella salina (Lamersa et al.
2012). Despite the decrease in PUFAs in ASW + P thalli as
compared with ASW + N and ASW + NP thalli, they showed
the highest content of C20 PUFAs (both AA and EPA), in
agreement with the earlier report of AA (C20:4, n6) accumulation in Parietochloris incisa under N starvation. In addition,
an increase in the availability of PUFAs in nutrient-supplemented thalli increased the LOX activity concomitant with
the increase in hydroxy oxylipin compounds that exhibit
defensive roles in combating oxidative stress conditions in
macroalgae (Kumar et al. 2010, Kumar et al. 2011). Further,
the decrease in hydroperoxy oxylipins was in agreement with
the earlier report of decreased lipid peroxidation (lipid hydroperoxide as well as TBARS-MDA levels) in nutrient-supplemented thalli in comparison with starved thalli (Kumari
et al. 2012). The anomaly of the highest hydroperoxy oxylipins
but lowest LOX activity found in ASW thalli may be explained
on the lines of non-enzymatic lipid peroxidation initiated by
ROS due to nutritional stress-induced oxidative stress in ASW
thalli, as observed in our earlier study (Kumari et al. 2012) and
also reported by Yu and Yang (2008). The increase in LOX
activity and the relative oxylipin contents of 9- and 13-HODE,
and 9-and 13-HOTrE suggested the up-regulation of 9-linoleate LOX and 13-linolentae LOX, respectively, in nutrientsupplemented thalli. There is only one report of the upregulation of the 13-LOX pathway in potassium deficiency
in A. thaliana, whereas no transcriptional responses were
found for nitrogen or phosphorus diminution (Troufflard
et al. 2010). Further, despite knowing the low content of
AA obtained in Ulva sp., LOX activity for AA substrate was
also investigated because this alga has earlier been reported to
show appreciable LOX activity with AA (Akakabe et al. 2002,
Kuo et al. 1997, Tsai et al. 2008). In our experience also, we
found 8-, 11-, 12- and 15-LOX isoforms in Ulva spp. with an
appreciable amount of HETEs (Kumari et al. 2013b). In the
present study, a 2.8- to 4.2-fold up-regulation of arachidonate
LOX activity was observed in nutrient-supplemented thalli
(Fig. 4), especially of arachidonate 15- and 5-LOX isoforms
in all the nutrient-supplemented thalli, and of arachidonate

Responses of Ulva lactuca under nutritional stress

Materials and Methods

Please see the supplementary data for a description of the
Materials and Methods.

Supplementary data
Supplementary data are available at PCP online.

Funding
This study was supported by the Council of Scientific and
Industrial Research (CSIR) [senior research fellowship (SRF)
CSIR Award No. 31/028(0101)/2009-EMR-1 to the first author
P.K.].

Acknowledgments
We would like to thank the Head, Analytical Sciences,
CSIR-CSMCRI for permitting us to use the GC facility.

Disclosures
The authors have no conflicts of interest to declare.

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the metabolic pathways of pigments and altering lipids, including a shift in lipid classes, FAs (unsaturated FA/SFA ratio) and
oxylipins. NL accumulation in starved conditions lags behind
carbohydrate synthesis, and 7 d of nutrient starvation was not
sufficient to shift carbon metabolism towards lipid biosynthesis.
Ulva lactuca accumulates DGDG, SQDG and DGTS when
deprived of either nitrate, phosphate or both, while supplementation of nutrients, especially nitrate, leads to retrieval of lost
MGDG. Similarly, PL synthesis is also dependent on phosphate
availability, while phosphate deprivation results in degradation
of PLs to PA or lyso-phospholipids. Further, the antagonistic
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us to hypothesize that lipidomic and biochemical changes in
U. lactuca could be regulated by IAA/kinetin cross-talk and
need further investigation.

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