cloning, characterization and functional analysis of...

Journal of Integrative Plant Biology 2013, 55 (6): 490–503

Research Article

Cloning, Characterization and Functional Analysis ofTwo Type 1 Diacylglycerol Acyltransferases (DGAT1s)from Tetraena mongolicaMinchun Li1,2, Mingming Zhao1,2, Hanying Wu1, Wang Wu1,2 and Yinong Xu1

∗

1Key Laboratory of Photobiology, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China2University of Chinese Academy of Sciences, Beijing 100049, China∗Corresponding author

Tel: +86 10 6283 6945; Fax: +86 10 6283 6504; E-mail: [email protected] online on 11 March 2013 at www.jipb.net and www.wileyonlinelibrary.com/journal/jipbdoi: 10.1111/jipb.12046

Abstract

Two cDNAs encoding putative type 1 acyl-CoA: diacylglycerol acyltransferases (DGAT1, EC 2.3.1.20),were cloned from Tetraena mongolica Maxim., an extreme xerophyte with high oil content in the stems.The 1,488-bp and 1,485-bp of the open reading frame (ORF) of the two cDNAs, designated as TmDGAT1aand TmDGAT1b, were both predicted to encode proteins of 495 and 494 amino acids, respectively.Southern blot analysis revealed that TmDGAT1a and TmDGAT1b both had low copy numbers in theT. mongolica genome. In addition to ubiquitous expression with different intensity in different tissues,including stems, leaves and roots, TmDGAT1a and TmDGAT1b, were found to be strongly induced byhigh salinity, drought and osmotic stress, resulting in a remarkable increase of triacylglycerol (TAG)accumulation in T. mongolica plantlets. TmDGAT1a and TmDGAT1b activities were confirmed in theyeast H1246 quadruple mutant (DGA1, LRO1, ARE1, ARE2) by restoring DGAT activity of the mutant hostto produce TAG. Overexpression of TmDGAT1a and TmDGAT1b in soybean hairy roots as well as inT. mongolica calli both resulted in an increase in oil content (ranging from 37% to 108%), accompaniedby altered fatty acid profiles.

Keywords: Diacylglycerol acyltransferase1; Tetraena mongolica; triacylglycerol.

Li M, Zhao M, Wu H, Wu W, Xu Y (2013) Cloning, characterization and functional analysis of two type 1 diacylglycerol acyltransferases (DGAT1s)from Tetraena mongolica. J. Integr. Plant Biol. 55(6), 490–503.

Introduction

Triacylglycerol (TAG), the main component of plant oil, rep-

resents a compact molecule for energy and carbon storage in

organisms because of its highly reduced state (Li-Beisson et al.

2010). Although TAG is typically accumulated in seeds, a range

of non-seed tissues including leaves, flower petals, anthers,

pollen grains and fruits are also capable of TAG synthesis (Lin

and Oliver 2008; Xu et al. 2008; Durrett et al. 2010). Besides

serving as a source of dietary calories for people, TAG from

plants is increasingly a source of renewable biomaterials and

fuels (Chapman and Ohlrogge 2012). Thus, any improvement

in TAG production is of interest and value, and plenty of

previous work has focused on this aim within the biotechnology

field (Andrianov et al. 2010; Taylor et al. 2010; Guihéneuf et al.

2011).

Triacylglycerol is synthesized by sequential transfer of fatty

acyl chains from acyl-CoA through the endoplasmic reticulum

(ER)-based Kennedy pathway (Kennedy 1961), which is con-

sidered to be the major pathway in TAG biosynthesis, although

acyl-CoA-independent pathways may also make marked con-

tributions to TAG formation, such as phosphatidylcholine: dia-

cylglycerol acyltransferase (PDAT), which uses phosphatidyl-

choline (PC) as the acyl donor (Dahlqvist et al. 2000).

Acyl-CoA: diacylglycerol acyltransferase (DGAT, EC

2.3.1.20), which is a sn-3-specific acyltransferase of DAG,mediates the final rate-limiting step of TAG biosynthesis in the

Kennedy pathway (Ohlrogge and Browse 1995; Dircks and

C© 2013 Institute of Botany, Chinese Academy of Sciences

Cloning and Functional Analysis of DGAT1 Genes 491

Sul 1999). Two sequence-unrelated classes of DGAT en-

zymes, DGAT1 and DGAT2, have been isolated from various

plants in previous research (Lardizabal et al. 2001; He et al.

2004; Kroon et al. 2006; Shockey et al. 2006; Xu et al. 2008;

Mañas-Fernández et al. 2009). DGAT1s are members of the

membrane-bound O-acyltransferase protein superfamily that

possess at least six transmembrane domains, while two to four

transmembrane domains were found in DGAT2 (Andreia et al.

2011). Moreover, DGAT1 and DGAT2 enzymes were shown

to localize to different subdomains of the ER in the tung tree

(Vernicia fordii) (Shockey et al. 2006), and were thus suggestedto play non-redundant roles in different tissues and species in

TAG synthesis (Shockey et al. 2006; Li et al. 2010). Over-

expression and mutational downregulation studies of DGAT1from Arabidopsis thaliana revealed that DGAT1 contributessignificantly to seed TAG biosynthesis (Zou et al. 1999; Jako

et al. 2001). Contributions of DGAT1 to TAG biosynthesis werealso confirmed by studies on soybean (Settlage et al. 1998),

maize (Zheng et al. 2008) and nasturtium (Xu et al. 2008).

The emerging role of DGAT2 orthologs appears to be more

important for the incorporation of unusual fatty acids in the seed

oils of some plants (Kroon et al. 2006; Shockey et al. 2006;

Burgal et al. 2008). A third soluble class of DGAT enzyme was

reported in peanut (Saha et al. 2006). In Arabidopsis, DGAT3is involved in a process of recycling linoleic (18:2) and linolenic

(18:3) fatty acids into TAG when seed oil breakdown is blocked,

and may play a key role in lipid metabolism (Hernández et al.

2012).

In addition to the above, a few workers have also docu-

mented that DGAT1 activity and its control over glycerolipid

assembly may be influenced by environmental factors. It was

demonstrated that an Arabidopsis mutant deficient in DGAT1displayed heightened sensitivity to abscisic acid (ABA) and

osmotic stress (Lu and Hills 2002), while Brassica napusoverexpressing DGAT1 led to more seed oil in the matureseed under drought conditions when compared to the control

(Weselake et al. 2008). These results indicate the potential

use of DGAT1 under stress conditions, and make studying the

manipulation of DGAT1 more attractive.

T. mongolica a member of the narrowly monotypic genusof Zygophyllaceae endemic to the western Gobi desert of

Inner Mongolia, China (Qian and Chen 2004), is an extreme

xerophyte with a well-developed root system, and can there-

fore tolerate drought and other harsh natural conditionsparticularly well (Zhang et al. 2003). Previous work showed

that T. mongolica can accumulate large amounts of TAG(approximately 46 mg/g of dry weight (DW)) in stem tissues

(Wang et al. 2007). Such a plant might be of potential value

to be used as an energy plant. In the present study, we

report the cloning, characterization, and functional analysis

of two DGAT1s from such a plant, in an attempt to pro-vide a possible means to affect changes in oil content, and

as a preliminary study for the exploitation and use of this

plant.

Results

TAG content and its fatty acid composition in roots,stems and leaves of T. mongolica plantlets

The TAG content in roots, stems, and leaves of T. mongolicaplantlets was determined by gas chromatography (GC) anal-

ysis. As shown in Table 1, TAG content in roots was the

highest at approximately 1.8 mg/g of DW, followed by stems

and then leaves. Table 1 also shows the fatty acid composition

of TAG from different tissues of T. mongolica plantlets. Theresults show that five common fatty acids, including palmitic

(16:0), stearic (18:0), oleic (18:1), 18:2 and 18:3, exist as the

main constituents. The fatty acid profiles of TAG from different

tissues of the plantlets were similar. The most prevalent fatty

acid was 18:3, which accounted for 30.6%–38.1% of the total

fatty acids in TAG. The polyunsaturated fatty acids (18:2 and

18:3) accounted for about 50% of the total TAG fatty acids.

The percentage of 16:0 was 22.7%–27.1% of the total TAG

fatty acids, which was second only to 18:3. Moreover, TAG

was relatively low in 18:0 and 18:1.

Isolation of two cDNA clones encoding DGAT1 fromT. mongolica plantlets

For the cloning of T. mongolica DGAT1, conserved regionsof DGAT1s were identified by alignment of deduced amino

acid sequences from different species. One DNA fragment

of 852-bp was amplified from T. mongolica plantlets using adegenerate primers approach, with the primers designed based

on DNA sequences of the conserved regions. However, this

852-bp fragment proved to be two different sequences after

sequencing. On the basis of the sequence information, gene-

specific primers for 3′- and 5′-RACE (rapid amplification ofcDNA ends) were generated, yielding two full-length cDNAs

named TmDGAT1a and TmDGAT1b, respectively.Sequence analysis indicates that TmDGAT1a (GenBank

accession No. JX163929) contains an ORF of 1,488-bp en-

coding a protein of 495 amino acids, and the 1,485-bp ORF

of TmDGAT1b (GenBank accession No. JX163930) was pre-dicted to encode a protein of 494 amino acids. The predicted

Mr and calculated isoelectric points are 57.1 kDa and 9.04for TmDGAT1a, and 56.8 kDa and 9.19 for TmDGAT1b (Prot-

param: http://www.expasy.ch), respectively. The two DGAT1s

share 82.9% similarity in nucleotide sequence and 82.7%

identity in deduced amino acid sequence. Phylogenetic tree

analysis of deduced amino acid sequences from TmDGAT1a

and TmDGAT1b and other known orthologs indicates that both

TmDGAT1a and TmDGAT1b are grouped together with all

492 Journal of Integrative Plant Biology Vol. 55 No. 6 2013

Table 1. Fatty acid composition of triacylglycerol (TAG) extracted from roots, stems and leaves of Tetraena mongolica plantlets

Fatty acid composition (mol%)

Sample TAG content (mg/g DW) 16:0 18:0 18:1 18:2 18:3

Roots 1.8 ± 0.1 24.4 ± 0.4 7.0 ± 0.6 19.1 ± 1.4 16.4 ± 1.0 33.1 ± 1.4Stems 1.3 ± 0.1 22.7 ± 1.1 6.6 ± 0.5 13.4 ± 0.9 26.7 ± 0.3 30.6 ± 1.4Leaves 1.2 ± 0.1 27.1 ± 1.8 7.9 ± 0.4 11.9 ± 1.1 15.0 ± 1.2 38.1 ± 1.2Data are the mean ± SD of three independent experiments.

known DGAT1s (Figure S1). Alignment of the deduced amino

acid sequences of TmDGAT1s and other DGAT1s from Ara-bidopsis, Nicotiana, Vernicia, maize and castor shows that theproteins share a high identity (44.3%, Figure 1). The C-terminal

regions (56.6% identity within 422 amino acids) are much more

conserved than the N-terminal regions (0.8% identity in the first

72 amino acids) (Figure 1).

Identification of putative functional motifs inTmDGAT1s

Scanning the amino acid sequences of both TmDGAT1a and

TmDGAT1b against the Prosite database (http://npsa-pbil.ibcp.

fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_proscan.html)

revealed a number of putative functional motifs shown in

Table S1. The quantity of some functional motifs in TmDGAT1a

and TmDGAT1b is different from AtDGAT1. It remains to be

determined whether these sites are important in the regulation

of the functions of the TmDGAT1s in vivo.In plants, DGAT1 has been localized to the ER membrane

where the Kennedy pathway mainly occurs (Settlage et al.

1995; Lacey and Hills 1996; De Domenico et al. 2011).

Nine potential transmembrane helices that likely anchor the

two proteins to the ER membrane were strongly predicted

by TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) for both

TmDGAT1a and TmDGAT1b (Figure S2). Little conservation

was found for the N-terminal amino acids, except for the

characteristic basic repeat consisting of three arginine residues

in TmDGAT1s, which is the same as that in other high

plants (Mañas-Fernández et al. 2009; Guihéneuf et al. 2011)

(Figure 1). Also detected was the presence of the reported acyl-

CoA binding signature (R83-G101 in TmDGAT1a, R82-G100

in TmDGAT1b) (Jako et al. 2001; Mañas-Fernández et al.

2009; Guihéneuf et al. 2011) close to residues (R116–N121

in TmDGAT1a, R115-N120 in TmDGAT1b) that have been

involved in the active site, as well as a DAG/phorbol es-

ter binding motif (Jako et al. 2001; Mañas-Fernández et al.

2009; Guihéneuf et al. 2011) (Figure 1). Within the pre-

viously reported leucine zipper motif (Lung and Weselake

2006; Mañas-Fernández et al. 2009; Guihéneuf et al. 2011),

an invariant proline residue (P191 in TmDGAT1a, P190 in

TmDGAT1b) (Figure 1) has been shown critical for DGAT1

activity (Xu et al. 2008). Another conserved feature is an

invariant serine among the shown DGAT1s at positions

220 and 219 in TmDGAT1a and TmDGAT1b, respectively

(Figure 1). The serine residue has been shown to be essential

for the activity of acyl-CoA: cholesterol acyltransferase, an en-

zyme closely related to DGAT1 (Joyce et al. 2000). In addition,

the leucine zipper motif was also found to overlap with a puta-

tive thiolase acyl-enzyme intermediate binding motif reported

for Arabidopsis, Tropaeolum and Phaeodactylum DGAT1 (Zouet al. 1999; Xu et al. 2008; Guihéneuf et al. 2011). There is

also a fatty acid-binding protein signature spanning residues

A356-N372 in TmDGAT1a and A355-N371 in TmDGAT1b,

which contains a putative tyrosine phosphorylation site, Y367

in TmDGAT1a and Y366 in TmDGAT1b (Xu et al. 2008)

(Figure 1). Two C-terminal motifs, YYHD-like and NRKG-like, as

the putative ER retrieval motifs, are also present in TmDGAT1s,

similar to other DGAT1 plants proteins (Lung and Weselake

2006) (Figure 1).

Gene structure and genomic organizationof TmDGAT1a and TmDGAT1b

A comparison between the partial genomic and cDNA se-

quences allows the identification of 15 introns interrupting the

coding region of TmDGAT1a and TmDGAT1b, respectively(Figure S3). To determine the copy number of the TmDGAT1sin the genome, Southern blot analysis was performed. For both

TmDGAT1a and TmDGAT1b, a single band was detected withthe restriction enzymes HindIII and XbaI (Figure S4, lanes2, 3, 5 and 6), and more than one band were detected with

EcoRI (Figure S4, lanes 1 and 4), probably due to the presenceof similar sequences of TmDGAT1s, because there was noEcoRI site in the probes used. These results indicate that bothTmDGAT1a and TmDGAT1b are with low copy number in thegenome of T. mongolica.

Tissue-specific expression of TmDGAT1s inT. mongolica plantlets

Quantitative real time PCR (qRT-PCR) was performed to

quantify TmDGAT1s transcripts in roots, stems and leavesof T. mongolica plantlets. The changes in expression levelsof TmDGAT1 transcripts in T. mongolica plantlets were de-termined relative to the expression level of the TmDGAT1a


Figure 1. Homology comparison of deduced amino acid sequences of TmDGAT1s with their orthologs from GenBank.

The alignment was generated with CLUSTAL W. VfDGAT1 (Vernicia fordii, Accession No. ABC94471.1), RcDGAT1 (Ricinus communis,

Accession No. XP002514132.1), AtDGAT1 (Arabidopsis thaliana, Accession No. NP179535.1), NtDGAT1 (Nicotiana tabacum, Accession

No. AAF19345.1), ZmDGAT1 (Zea mays, Accession No. ABV91586.1), TmDGAT1a and TmDGAT1b (Tetraena mongolica, Accession Nos.

JX163929 and JX163930). Conserved motifs or putative signatures (see text for details) are boxed, such as the N-terminal basic motif RRR

in higher plants (I), the acyl-CoA binding signature (II), the fatty acid protein signature (III), which contains a tyrosine phosphorylation site (�),the DAG-binding site (IV) and two C-terminal motifs –YYHD-like and NRKG-like (V) – as the putative endoplasmic reticulum retrieval motif

in the C-terminus. The region containing a conserved leucine repeat (L) in higher plants coincides with a thiolase acyl enzyme intermediate

binding signature (marked by a horizontal solid bar) beside the previously-described critical proline and serine residues, which are marked

by asterisks. The conserved phenylalanine is designated by an arrow.


transcript in the roots. The transcript abundance was normal-

ized to that of the TmACTIN gene. The two genes with differenttranscription profiles expressed in all tissues of T. mongolicaplantlets. We observed the highest steady-state level accu-

mulation of TmDGAT1b transcripts in roots and stems, whichwere much higher than that in leaves (Figure 2). Transcript

levels of TmDGAT1a in stems were higher than that in rootsand leaves, but were all at a much lower level compared to

TmDGAT1b (Figure 2). Taken together, these results suggestthat the expression level of TmDGAT1b is much higher thanthat of TmDGAT1a in the whole plant, particularly in the rootsof T. mongolica plantlets.

Expression patterns of TmDGAT1s in responseto NaCl, PEG and sucrose treatments

Given the fact that T. mongolica was able to survive harsh nat-ural conditions including drought, we first investigated whether

expression of TmDGAT1s is induced by stress treatments.One-m-old plantlets were treated with 800 mM NaCl, 20%

polyethylene glycol (PEG) and 15% sucrose, respectively,

and expression patterns of TmDGAT1s in root tissues wereanalyzed by qRT-PCR . The results indicated that there was a

time-dependent increase in both TmDGAT1a and TmDGAT1btranscripts upon exposure to 800 mM NaCl, 20% PEG and

15% sucrose treatments in roots of the T. mongolica plantlets(Figure 3A–C). As shown in Figure 3A, transcripts of TmDGAT1awere found to be accumulated after 2 h of NaCl treatment,

reached a maximum of 10-fold of the control at 24 h, and stayed

at this level thereafter. In contrast to TmDGAT1a, transcriptsof TmDGAT1b were found to have accumulated after 5 h ofexposure to salt stress, and they continued to increase for

at least 48 h, reaching a maximum of 5-fold of the control.

In addition to salt stress, the expression of TmDGAT1s was

Figure 2. Quantitative real time PCR (qRT-PCR) analysis of

TmDGAT1a and TmDGAT1b expression in different organs of

Tetraena mongolica plantlets.

Data are the mean ± SD of three independent experiments.

also upregulated by PEG-induced drought stress (Figure 3B).

TmDGAT1a transcripts began to accumulate after 1 h of treat-ment by 20% PEG, and exhibited a time-dependent increase,

reaching a maximum of 25-fold of the control at 48 h after start-

ing the treatment (Figure 3B). TmDGAT1b transcripts beganto accumulate after 5 h of treatment by PEG, and peaked at

24 h (Figure 3B). Moreover, the expression of TmDGAT1s wasalso induced by exposure to 15% sucrose, and TmDGAT1sexpression patterns were similar to those under PEG treatment

(Figure 3C). The results showed that expression of both

TmDGAT1a and TmDGAT1b was significantly upregulated byNaCl, PEG and sucrose treatment.

TAG accumulation in roots of T. mongolica plantlets inresponse to NaCl, PEG and sucrose treatments

Since the transcripts of TmDGAT1s were significantly in-creased by NaCl, PEG and sucrose treatment, TAG content

in roots of T. mongolica plantlets under stress were furtherexamined. As expected, oil deposition in roots of T. mongolicaplantlets exhibited a typical time-dependent increase pattern

under applied stress (Figure 3D–F). After exposure to 800 mM

NaCl for 2 h, a consistent increase in TAG accumulation in

roots of the plantlets was observed, which finally reached a

level 3-fold higher than that of the control at 72 h (Figure 3D).

TAG content increased much more slowly under 20% PEG

treatment, with the largest increase occurring during the last

24 h. The amount of TAG finally reached 7.73 mg/g of DW,

approximately 6-fold higher than that of the control (Figure 3E).

Similar to NaCl treatment, 15% sucrose treatment could also

result in an increase of TAG deposition in the roots of the

plantlets. However, TAG accumulation was found to increase

after 10 h of sucrose treatment – a delay of 8 h compared

with NaCl treatment (Figure 3F). The results indicated that the

increase of TAG content in the roots of plantlets might be mainly

due to the increase of the expression level of TmDGAT1sinduced by stress.

Heterologous expression of TmDGAT1a andTmDGAT1b in Saccharomyces cerevisiae

To assess TmDGAT1s activity, a complementation assay

was carried out using a Saccharomyces cerevisiae neu-tral lipid (NL)-deficient quadruple mutant strain (H1246)

(Sandager et al. 2002). Mutant strain H1246 transformed with

pYES2-TmDGAT1a or pYES2-TmDGAT1b was designated asTD1a/H1246 or TD1b/H1246, respectively. SCY62 (wild-type)

and H1246 cells harboring an empty pYES2 vector were used

as positive and negative controls, designated pYES2/SCY62

and pYES2/H1246, respectively. GC analysis showed that the

content of TAG extracted from TD1a/H1246 and TD1b/H1246

was approximately 18 mg/g and 20 mg/g, resulting in increases


Figure 3. Analysis of TmDGAT1s expression and triacylglycerol (TAG) content in roots of Tetraena mongolica plantlets in response

to applying stress.

(A) Quantitative real time PCR (qRT-PCR) analysis of TmDGAT1s expression in response to 800 mM NaCl treatment for varying periods.

(B) qRT-PCR analysis of TmDGAT1s expression in response to 20% PEG6000 treatment for varying periods.

(C) qRT-PCR analysis of TmDGAT1s expression in response to 15% sucrose treatment for varying periods.

(D) TAG content in roots of T. mongolica plantlets in response to 800 mM NaCl treatment for varying periods.

(E) TAG content in roots of T. mongolica plantlets in response to 20% PEG6000 treatment for varying periods.

(F) TAG content in roots of T. mongolica plantlets in response to 15% sucrose treatment for varying periods.

Data are the mean ± SD of three independent experiments. ∗∗P < 0.01


of 28% and 42%, respectively, compared to the positive control

(Figure 4). These results suggested that both TmDGAT1a andTmDGAT1b encode proteins with DGAT1 activities.

Expression of TmDGAT1s in Glycine max hairy roots

A non-seed system, Glycine max hairy root, was used to betterunderstand the function of TmDGAT1s. We generated thetransgenic G. max hairy roots by the overexpression constructspEGAD-TmDGAT1a and pEGAD-TmDGAT1b (Figure S5A).The phosphinothricin (PPT)-resistant hairy roots were exam-

ined by reverse transcription PCR (RT-PCR). Amplification with

primers for TmDGAT1a and TmDGAT1b, respectively, resultedin a single band with the expected size of approximately

1 500 bp (Figure S5B, C). Four transgenic hairy root lines were

maintained for TmDGAT1a (designated TD1a/HR-1, TD1a/HR-2, TD1a/HR-3 and TD1a/HR-4), and another four transgenic

hairy root lines were maintained for TmDGAT1b (designatedTD1b/HR-1, TD1b/HR-2, TD1b/HR-3, and TD1b/HR-4). Hairy

root lines obtained from the explants transformed by K599

containing empty pEGAD were used as the control for GC

analysis. As shown in Figure 5A and B, on a residue DW basis,

the transgenic hairy root lines of TmDGAT1a and TmDGAT1bboth exhibited an increase in TAG content, compared with

the control. Moreover, the increase of TAG content generally

ranged from 37% to 67% of TmDGAT1a transgenic lines, and

Figure 4. Gas chromatography (GC) analysis of triacylglycerol

(TAG) content obtained from yeast cells.

TAG was extracted from mutant strain H1246 transformed

by pYES2-TmDGAT1a (designated TD1a/H1246) or pYES2-

TmDGAT1b (designated TD1b/H1246). The wild-type strain SCY62

and the mutant strain H1246 harboring the empty plasmid were

used as positive and negative controls, designated pYES2/SCY62

and pYES2/H1246, respectively.

from 53% to 85% of TmDGAT1b transgenic lines. The fattyacid composition of TmDGAT1 transgenic hairy root lines wereboth significantly affected, with an increase of 19% to 37% for

18:2 as well as a decrease of 24% to 32% for 18:3 in TD1a/HRs

(Figure 5C), an increase of 22% to 44% for 18:2, and a decrease

of 12% to 23% for 18:3 in TD1b/HRs (Figure 5D). In addition,

all of the transgenic hairy root lines of TmDGAT1a exhibited anincrease of 15% to 33% for 16:0, compared with the control

(Figure 5C). The results indicate that both TmDGAT1a andTmDGAT1b are functional in G. max hairy roots. Besides anincrease in TAG content, there was also an alteration of fatty

acid composition in the transgenic lines of both TmDGAT1aand TmDGAT1b.

Expression of TmDGAT1s in T. mongolica calli

T. mongolica calli cells were also used to further verify the func-tions of the two genes. The pEGAD-TmDGAT1a and pEGAD-TmDGAT1b constructs were introduced into Agrobacteriumtumefaciens EHA105 and used to transform T. mongolica callicells induced from stems of plantlets, and the progeny were

analyzed as described in the Materials and Methods section

of this paper. TmDGAT1s-overexpressed calli generated byPPT selection and confirmed by RT-PCR for the presence

of Bar gene (data not shown) were obtained and namedTD1a/TmC and TD1b/TmC, respectively. qRT-PCR analysis

indicated that the expression of TmDGAT1a in TD1a/TmCincreased by 77%, and TmDGAT1b in TD1b/TmC increased by151%, compared to the control (Figure 6A,B). On a residue DW

basis, the TAG content of TD1a/TmC and TD1b/TmC increased

by 59% and 108%, respectively, compared to the control

(Figure 6C). The fatty acid composition of the TAG in transgenic

calli was also significantly affected by overexpression of the

TmDGAT1s. In TD1a/TmC, there was a significant increasein 18:2 of 80%, as well as a decrease of 25% in 18:3. 18:1

was also greatly enriched with an increase of 57%. Similar

changes of the fatty acid profiles also occurred in TD1b/TmC;

18:1 and 18:2 increased by 100% and 92%, respectively. In

addition, 18:3 decreased by 30%. Slight changes were also

observed in 16:0 and 18:0 in both TD1a/TmC and TD1b/TmC

(Figure 6D).

Discussion

We cloned and characterized two DGAT1 genes (TmDGAT1aand TmDGAT1b) from T. mongolica, which encode pro-teins with high homologies and significant primary structural

similarities to other previously identified DGAT1 genes in dif-ferent plant species (Figure 1, Figure S1). Protein hydrophobic-

ity analysis predicted that both TmDGAT1a and TmDGAT1b

possess nine putative transmembrane domains, a structural

feature, which correlates with other plant DGAT1s and is


Figure 5. Gas chromatography (GC) analysis of triacylglycerol (TAG) content in transgenic soybean hairy roots of TmDGAT1s.

(A) TAG content of transgenic hairy root lines of TmDGAT1a.

(B) TAG content of transgenic hairy root lines of TmDGAT1b.

(C) Fatty acid compositions of TAG extracted from transgenic hairy root lines of TmDGAT1a.

(D) Fatty acid compositions of TAG extracted from transgenic hairy root lines of TmDGAT1b.

TD1a/HR, transgenic hairy roots of TmDGAT1a; TD1b/HR, transgenic hairy roots of TmDGAT1b; control, hairy roots containing empty

pEGAD. Data are the mean ± SD of three independent experiments. ∗∗P < 0.01

consistent with its role as an integral membrane protein that

has been shown to be localized to the ER (Zou et al. 1999;

Nykiforuk et al. 2002; He et al. 2004; Shockey et al. 2006).

Southern blot analysis indicated that both TmDGAT1a andTmDGAT1b were with low copy number in the T. mongolicagenome (Figure S4). Quantification of TmDGAT1s transcriptsby qRT-PCR showed that both TmDGAT1a and TmDGAT1bubiquitously expressed in all tissues of T. mongolica plantlets(Figure 2), which is the same situation as in A. thaliana DGAT1(Zou et al. 1999). TmDGAT1b transcript intensity was muchstronger than TmDGAT1a in all tissues, and particularly inroots, which correlates quite well with TAG content from dif-

ferent tissues (Figure 2; Table 1). These results indicate that

TAG accumulation in different tissues of T. mongolica plantletsmay largely be due to TmDGAT1b.

However, TAG content reported here was much lower than

that of T. mongolica growing in the local environment (Wanget al. 2007). The TAG content difference may be due to different

periods the plants were in and different environments in which

they survived. T. mongolica is a typical xerophytic plant inInner Mongolia, and could not survive successfully outside

its local environment, which makes it unqualified for study in

the laboratory. Therefore, we had to choose tissue culture

plantlets as research materials in this paper. Normally, TAG

is an efficient high density carbon and energy source that is

important in transferring reduced carbon from one generation

to the next. Thus, it is found mainly in seeds, pericarps and

pollen (Lin and Oliver 2008). T. mongolica plantlets from tissuecultures had higher water content and a lower lignified degree,

and were in a strong vegetative growth stage, which was not an

ideal storage mode for large amounts of TAG, compared with

the woody stems of the shrub. Wang et al. (2007) conjectured

that high levels of TAG in stems are advantageous to survival in

harsh natural surroundings, especially drought. However, the

plantlets used in this study were cultured in the greenhouse

where conditions are stable and unchallenged. It seems that

there is no need for the plantlets to accumulate large amounts

of TAG under such conditions. When the plantlets were treated

by applying stress, including high salinity, drought and osmotic

stress; TAG accumulation in T. mongolica plantlets exhibited atypical time-dependent increase pattern (Figure 3D–F), provid-

ing optimal adaptation to environmental factors. An increased


Figure 6. Analysis of TmDGAT1s expression and triacylglycerol (TAG) content in transgenic Tetraena mongolica calli.

(A) Quantitative real time PCR (qRT-PCR) analysis of TmDGAT1a expression in transgenic calli of TmDGAT1a.

(B) qRT-PCR analysis of TmDGAT1b expression in transgenic calli of TmDGAT1b.

(C) Analysis of TAG content in transgenic calli.

(D) Fatty acid compositions of TAG extracted from transgenic calli.

TD1a/TmC, transgenic T. mongolica calli of TmDGAT1a; TD1b/TmC, transgenic T. mongolica calli of TmDGAT1b; control, T. mongolica calli

containing empty pEGAD.

Data are the mean ± SD of three independent experiments. ∗∗P < 0.01

expression level of both TmDGAT1a and TmDGAT1b couldthus provide an explanation, at the molecular level, for the rapid

induction of TAG biosynthesis in T. mongolica plantlets underapplied stress (Figure 3A–C). Our results are consistent with

the fact that DGAT1 activity and its control over TAG assembly

may be influenced by environmental factors (Weselake et al.

2008). Similar to microalgae, TAG is mainly accumulated under

stressful conditions in extra-plastidial oil bodies (Guihéneuf

et al. 2011). The main work to be done in the future is to

investigate the molecular mechanism of large amounts of TAG

accumulation in stems in order to exploit and use this plant.

Results from heterologous expression studies on the

S. cerevisiae NL-deficient quadruple mutant confirmed thatthe TmDGAT1 genes encode proteins that function as DGAT1enzymes. When the recombinant TmDGAT1a and TmDGAT1b

proteins were expressed in the NL-deficient mutant strain

H1246 devoid of all enzymes, which can contribute to TAG

synthesis, it was shown that DGAT activity of the mutant host

was restored to produce TAG (Figure 4). In plant transformation

studies, T. mongolica calli and soybean hairy roots were usedas explants for further functional verification of TmDGAT1a andTmDGAT1b in this study. Overexpression of TmDGAT1a andTmDGAT1b in soybean hairy roots resulted in an increase inoil content of 37% to 85% (Figure 5A, B). Similar experiments

conducted using TmDGAT1a and TmDGAT1b overexpressionin T. mongolica calli showed that transgenic calli exhibited anoil content increase of 59% and 108%, respectively (Figure 6C).

These results further confirmed that both TmDGAT1a andTmDGAT1b encode proteins to function as DGAT1 enzymes. Inaddition, our results also support earlier biochemical evidence

indicating that DGAT is rate-limiting for the production of TAG

(Perry et al. 1999; Lung and Weselake 2006).

Besides an increase in oil content, the fatty acid composition

of TAG was also significantly affected in all transgenic T. mon-golica calli and transgenic hairy root lines by overexpression ofTmDGAT1a and TmDGAT1b, with an increase of 18:2 and 16:0as well as a dramatic decrease of 18:3 (Figure 5C,D; Figure 6D).

Moreover, transgenic T. mongolica calli also exhibited an


increase of 18:1 concomitant with a decrease of 18:0 in TAG

compared to the control (Figure 6D). The observed changes in

overexpression of TmDGAT1s are not very surprising. Similarresults have been obtained before. For overexpression of

Arabidopsis DGAT1 in tobacco, the percent of 18:3 in leafTAG was reduced from 20% to 12%, and 18:1 increased from

18% in wild-type (WT) to 44% in the analyzed transgenic line

(Andrianov et al. 2010). Even more profound, a single amino

acid insertion in DGAT resulted in significantly increased oil

and oleic acid content in maize seeds (Zheng et al. 2008).

DGATs typically exhibit a strong capacity for incorporating the

most prevalent acyl moieties into the sn-3 position (Xu et al.2008). Evidence from this work indicated that there seems

to be more 18:1 and 18:2 in the T. mongolica calli as wellas in soybean hairy roots. In addition, both TmDGAT1a and

TmDGAT1b appeared to prefer 18:1 and 18:2 rather than 18:0

and 18:3. Therefore, manipulation of the expression of these

two genes may both increase oil content and alter fatty acid

composition.

In the present study, transgenic soybean hairy roots and

transgenic T. mongolica calli were used as two quick andefficient systems for TmDGAT1s function verification, whichoffer rapid techniques to introduce foreign genes in plant cells

(Aarrouf et al. 2011). Production of transgenic hairy roots with

A. rhizogenes only takes a few weeks (Guillon et al. 2006).Genetically engineered root cultures have been used as a

model system to modulate the metabolism and regulation of

several natural product pathways (Zhang et al. 2009; Park

et al. 2011). Similar to soybean hairy roots, it also took only

a few weeks to harvest transgenic calli. In many high plant

species, calli were widely used as explants for plant trans-

formation and regeneration of transgenic plantlets because

of their high efficiency, including such species as Ponkanmandarin (Li et al. 2002) and apple trees (Dong et al. 2011).Transgenic calli could be preserved over the long-term, and

be induced to regenerate transgenic plants when necessary.

Moreover, the genetically identical background of the material,

easy proliferation and rare occurrence of chimeras during

regeneration are other positive features of this transformation

system. In this paper, the two systems were used for the first

time in studying DGAT1 functions, and performed very well inboth transformation and TmDGAT1s functional identification.

Materials and Methods

Plant materials and growth conditions

Tetraena mongolica Maxim. seeds were collected from severalisolated populations in the Wuhai Conservation Zone of T. mon-golica, Inner Mongolia, China, and were grown on MS medium(Murashige and Skoog 1962) after being sterilized in 70% (v/v)

alcohol for 3 min followed by a second sterilization in 10%

(w/v) NaClO for 15 min. All of the T. mongolica plantlets usedin this work were regenerated by stem segments cut from 2-

month-old seedlings and cultured on 1/2 MS medium containing

1 mg/L indolebutyric acid. After root formation, plantlets were

transferred to containers filled with vermiculite containing MS

liquid medium for subsequent growth. All media had 7 g/L agar,

and the pH was adjusted to 5.8 using 1 N NaOH or HCl, before

autoclaving at 121 ◦C, 105 kPa for 20 min. Cultures wereincubated at 26 ◦C ± 2 ◦C, 100 µmol/m2 per s, with a 16 hphotoperiod.

Stems of T. mongolica plantlets were used to establishT. mongolica calli on modified MS medium containing 0.5 mg/L2,4-D and 0.1 mg/L 6-BA, and were incubated at 26◦C ± 2 ◦C,100 µmol/m2 per s, with a 16-h photoperiod. The cultures were

sub-cultured every 28 d and used for experiments 20–25 d after

sub-culturing.

Soybean (Glycine max (L.) Merrill, cv. Heinong44) seedswere surface-sterilized in a hermetically sealed desiccator with

chlorine gas produced by mixing 100 mL bleach (5.25% sodium

hypochlorite) and 4 mL 12 N HCl for 14 h. Sterilized seeds were

germinated on MS medium containing 3% sucrose and 0.8%

agar at 26 ± 2 ◦C, 100 µmol/m2 per s, with a 16 h photoperiod.

Cloning of T. mongolica DGAT1 cDNAs

Degenerate primers were designed according to amino acid

sequences conserved in Arabidopsis and other known plantDGAT1s. Total RNA was isolated from 1-month-old T. mon-golica plantlets with RNAiso Plus (TaKaRa, Dalian, LiaoNing,China), and treated with RNase free DNase I (TaKaRa). A

single-stranded cDNA template for RT-PCR was synthesized

at 42 ◦C from plantlets poly (A) RNA using M-MLV reversetranscriptase (Promega, Madison, WI, USA). A 50 µL PCR

contained single-stranded cDNA derived from 40 ng of poly (A)

RNA, 12.5 nmol of each primer (DRf and DRr, see Table S2),

and 2.5 U of TransTaq DNA Polymerase High Fidelity (HiFi)(TransGen Biotech, Beijing, China) under standard conditions.

One internal part of the DGAT sequence was amplified in a

thermocycler for 30 cycles of the following program: 94 ◦C for30 s, 55 ◦C for 30 s and 72 ◦C for 1 min. The sequence of a852-bp PCR product was used to design gene-specific primers

(GSPs) to amplify the 5′ and 3′ ends of the cDNA as de-scribed by Scotto-Lavino et al. (2006, 2007), and finally yielded

two full-length cDNAs named TmDGAT1a and TmDGAT1b,respectively. The GSPs used for RACE are displayed in

Table S2.

Southern blot analysis

Genomic DNA was isolated from T. mongolica plantlets usinga modified CTAB-based extraction procedure (Murray and

Thompson 1980). Southern blot analysis was conducted ac-

cording to the methods described by Sambrook and Russell


(2001). Aliquots of genomic DNA (10 µg) were digested

overnight with three restriction enzymes, HindIII, EcoRI andXbaI, individually. Digoxigenin (DIG)-labeled probes were gen-erated by PCR spanning 291-bp of the 5′ coding sequenceof TmDGAT1a, and 288-bp of the 5′ coding sequence ofTmDGAT1b, and the corresponding primers used are displayedin Table S2.

Stress treatments of T. mongolica plantlets

For the treatment of high salinity, drought and osmotic stresses,

one-month-old plantlets grown in vermiculite containing MS

liquid medium were transferred to vermiculite watered with MS

liquid medium supplemented with 800 mM NaCl or 20% PEG

6000 or 15% sucrose, and roots of the plantlets were sampled

at 0.5 h (used as control), 1 h, 2 h, 5 h, 10 h, 24 h, 48 h and

72 h for analysis of gene expression and TAG content.

RNA isolation and quantitative PCR

Total RNAs were extracted from root tissues of treated T.mongolica plantlets with RNAiso Plus (TaKaRa), and treatedwith RNase free DNase I (TaKaRa). The total RNAs were

reverse-transcribed into first-strand cDNA in a 20-µL volume

with M-MLV reverse transcriptase (Promega). The samples

were diluted to 100 µL with water and 1 µL of each sample

(∼10 ng RNA equivalent) was amplified using TransStart GreenqPCR SuperMix UDG (TransGen Biotech) in a 20 µL reaction,

containing 1 µL of diluted cDNA, 10 µL TransStart Green qPCRSuperMix UDG, 0.4 µL Passive Reference Dye II, 1 µL of 10 µM

forward primer, 1 µL of 10 µM reverse primer and 6.6 µL of

water. The thermal cycle used was as follows: 50 ◦C for 2 min;95 ◦C for 10 min; 40 cycles of 95 ◦C for 5 s, 57 ◦C for 15 s and72 ◦C for 10 s. The corresponding specific primers are dis-played in Table S2. The relative expression levels were deter-

mined as described by Livak and Schmittgen (2001).

Expression of TmDGAT1s in yeast

Saccharomyces cerevisiae strains SCY62 (Relevant geno-type: MATaADE2), H1246 (Relevant genotype: MATαare1-�::HIS3 are2-�::LEU2 dga1-�::KanMX4 Iro1-�::TRP1ADE2)(Sandager et al. 2002) and the pYES2 expression vector were

kindly provided by S. Stymne (Scandinavian Biotechnology

Research, Alnarp, Sweden).

The full-length cDNA of TmDGAT1s was amplified by PCRwith the corresponding primers (see Table S2), and subse-

quently cloned behind the GAL1 inducible promoter in therespective sites of the pYES2 vector to create the pYES2-

TmDGAT1a and pYES2-TmDGAT1b constructs. The resultingplasmids were transformed to H1246 strain cells according to

the methods described by Elble (1992). H1246 and SCY62 cells

harboring the empty pYES2 vector were used as negative and

positive controls, respectively. Transformants were selected by

growth on synthetic complete medium lacking uracil (SC-ura),

supplemented with 2% (w/v) glucose.

For functional expression, the colonies were transferred into

liquid SC-ura with 2% (w/v) glucose and grown at 30 ◦Covernight. The overnight cultures were diluted to an OD 600

of 0.2 in induction medium (SC-ura + 2% galactose), and wereinduced for 36 h at 30 ◦C. Cells were collected for lipid analysis.

Plant transformation vectors

The full-length ORF of TmDGAT1s was amplified by PCRwith the corresponding primers (see Table S2), and subse-

quently cloned behind the cauliflower mosaic virus (CaMV)

35S promoter in the respective sites of the pEGAD vector

to create the pEGAD-TmDGAT1a and pEGAD-TmDGAT1bconstructs.

Plant transformation and selection

The pEGAD-TmDGAT1a and pEGAD-TmDGAT1b constructswere introduced into T. mongolica callus cultures using A.tumefaciens strain EHA105, as reported by Li et al. (2002).After 3 d of co-culture in darkness at 22 ◦C, the calli werewashed three to four times, and then transferred to selection

medium (MS medium containing 0.1 mg/L 6-BA, 0.25 mg/L

2, 4-D, 5 mg/L PPT and 500 mg/L carbenicillin). Four weeks

later, most of the calli had become necrotic and died. The calli

that did proliferate on the selection medium were sub-cultured

on selection medium three or four times at 3 week intervals.

Transgenic calli were further confirmed by PCR amplification of

cDNA using primers of Bar gene (see Table S2), and were thenused for further expression of TmDGAT1s and TAG analysis.

Agrobacterium rhizogenes strain K599 with binary vectorpEGAD-TmDGAT1a or the pEGAD-TmDGAT1b construct wasused to obtain transgenic hairy roots according to Cho et al.

(2000). Established root cultures were further detected by PCR

analysis of cDNA (primers used are displayed in Table S2), and

then used for further TAG analysis.

Lipid extraction and separation

The collected yeast cells were broken in a glass-bead shaker,

and extracted into chloroform: MeOH (1:2, v/v). Other sam-

ples, including different tissues separated from T. mongolicaplantlets, T. mongolica calli, and soybean hairy roots, wereground to a fine powder under liquid nitrogen, and subsequently

dumped into chloroform: MeOH (1:2, v/v) immediately. Total

lipids were extracted from the samples by methods described

by Bligh and Dyer (1959), and TAG was separated from the

total lipids by thin-layer chromatography (TLC). The plates

were developed in hexane: Et2O: AcOH (70:30:1, v/v). Spots


were made visible by spraying the plates with 0.01% primuline

in acetone/H2O (60:40; v/v) and examining the plates under

ultraviolet (360 nm) light. Triolein was used as the standard.

Residue of the samples after extracting was rinsed five times

with distilled water and dried at 80 ◦C for 24 h to determine theDW of residue.

Fatty acid analysis

Fatty acid analysis was carried out following the method

described by Xu et al. (2003). Briefly, the TAGs separated

by TLC were transesterified with 5% H2SO4 in MeOH at

85 ◦C for 1 h, and the fatty acid methyl esters (FAMEs) wereextracted with hexane and separated on a Hewlett-Packard

6890 gas chromatography apparatus supplied with a hydrogen

flame ionization detector and a capillary column HP INNOWAX

(30 m; 0.25 mm i.d.) with N2 carrier at 20 mL/min. The oven

temperature was maintained at 170 ◦C for 5 min, and thenincreased in steps to 210 ◦C, with the temperature beingraised by 5 ◦C every min. FAMEs from TAG were identifiedby comparing their retention times with known standards (37-

component FAME mix, Supelco47885-U). Heptacanoic acid

(17:0, from Sigma) served as the internal standard to quantify

the amounts of TAG.

Acknowledgements

This work was supported by the National Natural Science Foun-dation of China (30770224) and the National Basic ResearchProgram of China (2011CBA00901).

Received 1 Nov. 2012 Accepted 9 Feb. 2013

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Supporting Information

Additional Supporting Information may be found in the online

version of this article:

Figure S1. A phylogram showing relationships amongTmDGAT1s and DGAT1 proteins from other plants.The alignment was generated with CLUSTAL W, and the

phylogram was constructed by the neighbor-joining method

with MEGA.4: OeDGAT1 (Olea europaea, Accession no.AAS01606.1), PfDGAT1 (Perilla frutescens, Accession no.AAG23696.1), NtDGAT1 (Nicotiana tabacum, Accession no.AAF19345.1), GmDGAT1a and GmDGAT1b (Glycine max,Accession nos. AAS78662.1 and BAE93461.1), LjDGAT1

(Lotus japonica, Accession no. AAW51456.1), EaDGAT1(Euonymus alatus, Accession no. AAV31083.1), JcDGAT1(Jatropha curcas, Accession no. ABB84383.1), VfDGAT1 (Ver-nicia fordii, Accession no. ABC94471.1), RcDGAT1 (Ricinuscommunis, Accession no. XP002514132.1), BjDGAT1a andBjDGAT1b (Brassica juncea, Accession nos. AAY40784.1and AAY40785.1), AtDGAT1 (Arabidopsis thaliana, Acces-

sion no. NP179535.1),TmDGAT1 (Tropaeolum majus, Acces-sion no. AAM03340.2), ZmDGAT1 (Zea mays, Accessionno. ABV91586.1), OsDGAT1 (Oryza sativa, Accessionno. NP001054869.2), SmDGAT1 (Selaginella moellendorf-fii hypothetical protein, Accession no. XP002994237.1),VgDGAT1a and VgDGAT1b (Vernonia galamensis, Ac-cession nos. ABV21945.1 and ABV21946.1), EpDGAT1

(Echium pitardii, Accession no. ACO55634.1), TmDGAT1a andTmDGAT1b (Tetraena mongolica, Accession nos. JX163929and JX163930).

Figure S2. Putative transmembrane domains ofTmDGAT1a and TmDGAT1b.The main transmembrane segments were predicted by

TMHMM (http://www.cbs.dtu.dk/services/TMHMM/). Nine

predicted transmembrane helices were identified for both

TmDGAT1a and TmDGAT1b.

Figure S3. Genomic structure of TmDGAT1s.(A) Genomic structure of TmDGAT1a.(B) Genomic structure of TmDGAT1b.An examination of partial genomic clone of TmDGAT1s (8 043bp for TmDGAT1a and 7 588 bp for TmDGAT1b) allows theidentification of 15 introns interrupting the corresponding coding

region, respectively.Figure S4. Southern blot analysis of Tetraena mongolicagenomic DNA.(A) Analysis of the genomic organization of TmDGAT1a bySouthern blot.

(B) Analysis of the genomic organization of TmDGAT1b bySouthern blot.

T. mongolica genomic DNA (10 ug/lane) was digested withrestriction enzymes EcoRI (lane 1 and lane 4), HindIII (lane 2and lane 5) and XbaI (lane 3 and lane 6). The DNA blot washybridized with a dioxigenin-labeled partial cds of TmDGAT1aor TmDGAT1b as a probe, respectively. Marker sizes (Kb) areindicated.

Figure S5. Induction and examination of soybean hairyroots.(A) Induction of hairy roots on soybean cotyledons 3 weeks af-ter infection with A. rhizogenes K599 containing empty pEGADor pEGAD-TmDGAT1a or pEGAD-TmDGAT1b.(B) The PPT resistant hairy roots were examined by RT-PCRwith primers responsible for TmDGAT1a. Lane 2 to lane 5,transgenic lines of TmDGAT1a; lane 1, negative control; Lane6, positive control.

(C) The PPT resistant hairy roots were examined by RT-PCRwith primers responsible for TmDGAT1b. Lane 8 to lane 11,transgenic lines of TmDGAT1b; lane 7, negative control; lane12, positive control.

M, Marker.

Table S1. Putative functional motifs in TmDGAT1s andAtDGAT1Table S2. A list of oligonucleotides used in this study

cloning, characterization and functional analysis of...

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