Constitutive or seed-specific overexpression of Arabidopsis G-protein γ subunit 3 ( AGG3 ) results...

Constitutive or seed-specific overexpression ofArabidopsis G-protein c subunit 3 (AGG3) resultsin increased seed and oil production and improvedstress tolerance in Camelina sativaSwarup Roy Choudhury, Adam J. Riesselman and Sona Pandey*

Donald Danforth Plant Science Center, St. Louis, MO, USA

Received 25 April 2013;

revised 25 July 2013;

accepted 27 July 2013.

*Correspondence (Tel +314 587 1471;

fax +314 587 1571;

email [email protected])

Keywords: Camelina sativa,

heterotrimeric G-proteins, AGG3, seed

number and size, stress tolerance, oil

content.

SummaryHeterotrimeric G-proteins consisting of Ga, Gb and Gc subunits play an integral role in mediating

multiple signalling pathways in plants. A novel, recently identified plant-specific Gc protein,

AGG3, has been proposed to be an important regulator of organ size and mediator of stress

responses in Arabidopsis, whereas its potential homologs in rice are major quantitative trait loci

for seed size and panicle branching. To evaluate the role of AGG3 towards seed and oil yield

improvement, the gene was overexpressed in Camelina sativa, an oilseed crop of the

Brassicaceae family. Analysis of multiple homozygous T4 transgenic Camelina lines showed that

constitutive overexpression of AGG3 resulted in faster vegetative as well as reproductive growth

accompanied by an increase in photosynthetic efficiency. Moreover, when expressed

constitutively or specifically in seed tissue, AGG3 was found to increase seed size, seed mass and

seed number per plant by 15%–40%, effectively resulting in significantly higher oil yield per

plant. AGG3 overexpressing Camelina plants also exhibited improved stress tolerance. These

observations draw a strong link between the roles of AGG3 in regulating two critical yield

parameters, seed traits and plant stress responses, and reveal an effective biotechnological tool

to dramatically increase yield in agricultural crops.

Introduction

Heterotrimeric GTP-binding proteins (G-proteins hereafter) are

important regulators of multiple growth and developmental

pathways in eukaryotes. G-protein complex, consisting of Ga, Gband Gc subunits, switches between active and inactive confor-

mation depending on the guanine nucleotide-bound status of Gaprotein. GDP-Gabc trimeric complex represents the inactive state

of signalling. A signal-dependent exchange of GDP for GTP on Garesults in the formation of GTP-Ga and freed Gbc. Both these

entities can transduce the signal by interacting with various

intracellular effectors. The intrinsic GTPase activity of Ga causes

production of GDP-Ga, which re-associates with the Gbc to

return to the GDP-Gabc conformation (Cabrera-Vera et al.,

2003; Offermanns, 2003). In plants, the involvement of

G-proteins has been established in the regulation of a multitude

of fundamental growth and development pathways especially

phytohormone signalling and cross-talk, cell division, ion channel

activity and defence responses (Urano et al., 2013).

Although evolutionarily conserved, plants contain fewer

G-proteins compared with metazoans. While 23 Ga, 5 Gb and

12 Gc subunits are present in humans, the model plant

Arabidopsis thaliana has only one Ga, one Gb and three Gc-proteins (Temple and Jones, 2007). In this plant, the specificity of

heterotrimer formation is thus solely provided by the Gc proteins.

The plant Gc proteins are fairly diverse and classified into three

different subtypes based on their structural features: types I, II and

III (Roy Choudhury et al., 2011). The type I and type II families

exhibit most of the conserved features of canonical Gc proteins.

The type III Gc proteins, represented by AGG3 in Arabidopsis and

GmGc8, GmGc9 and GmGc10 in soybean, are recently discov-

ered novel, plant-specific proteins that are almost twice as large

as typical Gc proteins (Chakravorty et al., 2011; Li et al., 2012;

Roy Choudhury et al., 2011). The N-terminal half of these

proteins exhibits a high degree of similarity with canonical Gcproteins, whereas the C-terminal half (70–140 amino acids) is

plant specific and contains an extremely high number of cysteine

residues. Functional analysis of Arabidopsis AGG3 shows its

involvement in G-protein-mediated abscisic acid (ABA) signalling

pathways (Chakravorty et al., 2011). Similarly, in soybean, the

type III Gc proteins are involved during ABA-dependent inhibition

of nodule formation and lateral root development in transgenic

soybean hairy roots (Roy Choudhury and Pandey, 2013). In

addition, a novel role for the group III Gc proteins emerged in the

control of organ size and architecture based on the phenotypes of

multiple rice mutants. Two previously identified quantitative trait

loci (QTLs) for seed size and number, DEP1 (dense and erect

panicle 1) and GS3 (grain size 3), encode for possible homologs of

type III Gc proteins (Fan et al., 2009; Huang et al., 2009; Mao

et al., 2010; Takano-Kai et al., 2009). Targeted knockout and

overexpression of AGG3 gene in Arabidopsis support its role in

the regulation of organ size. The agg3 knockout mutants have

relatively smaller and fewer seeds per silique, whereas Arabidop-

sis plants overexpressing this gene have slightly larger and more

seeds per plant (Chakravorty et al., 2011; Li et al., 2012).

The regulation of two important agronomic traits, seed size

and number, as well as ABA responses by the type III Gc proteins

makes them a key target for potential biotechnological applica-

ª 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd 49

Plant Biotechnology Journal (2014) 12, pp. 49–59 doi: 10.1111/pbi.12115

tions. However, the exceptionally small size of Arabidopsis seeds

and their extreme dependence on the health of the maternal

plant and on specific growth conditions, together with the

relatively modest phenotypes of transgenic plants, confound the

impact of such observations. Furthermore, it remains to be

evaluated whether the overexpression of AGG3 has a positive

effect on the regulation of stress responses in plants even though

the agg3 mutants show altered ABA sensitivities (Chakravorty

et al., 2011). In addition, significant differences exist between the

phenotypes of G-protein mutants in Arabidopsis and other plant

species. RNAi-mediated suppression of G-protein a and bsubunits in rice results in dwarf plants that exhibit severe

morphological differences compared with the wild-type plants,

whereas transcript-null mutants of Arabidopsis G-protein a and bsubunits are not dwarf and exhibit relatively normal morphology

(Ueguchi-Tanaka et al., 2000; Utsunomiya et al., 2011). Similarly,

tobacco Gb-RNAi plants show differences in their pollen and

anther development, which are phenotypes not observed in

Arabidopsis agb1 plants (Peskan-Berghofer et al., 2005). There-

fore, further experimental evidence in additional plant systems is

required to establish the roles of G-proteins as universal regula-

tors of important yield parameters.

Camelina sativa (Camelina), an oilseed crop, is a close relative

of A. thaliana and has generated a great renewed interest as a

potential biofuel option (Ghamkhar et al., 2010; Nguyen et al.,

2013). Camelina is a low-input crop; it is relatively tolerant to

drought- , salinity- and frost-related stress compared with other

oilseed plants and grows well in low-nutrient soil (Carmo-Silva

and Salvucci, 2012; Ghamkhar et al., 2010; Kim et al., 2013).

Moreover, Camelina is genetically tractable, unlike many other

biofuel crops. It has a short lifecycle, can be easily transformed

using Agrobacterium-mediated floral dip method and produces

large quantities of seeds (Lu and Kang, 2008). Camelina genome

has recently been sequenced, and it shows a high degree of

sequence homology with Arabidopsis genes (Liang et al., 2013;

Nguyen et al., 2013). More importantly, Arabidopsis genes have

been shown to be functional in Camelina (Zhang et al., 2012).

Given the importance of Camelina as a significant biofuel source

and the potential of Arabidopsis AGG3 gene to improve seed-

related traits, biomass production and stress responsiveness, we

evaluated the effect of AGG3 overexpression in Camelina using a

constitutive (CaMV35S) and a seed-specific (soybean glycinin)

promoter. Our data show that AGG3 is a key positive regulator of

overall biomass production as well as seed size and number,

resulting in higher seed and oil yield in addition to providing

better stress tolerance.

Results

Generation of Camelina plants overexpressingArabidopsis AGG3

Arabidopsis AGG3 (AT5G20635) is a novel Gc protein involved in

guard cell K+-channel regulation, morphological development

and control of organ shape and size (Chakravorty et al., 2011; Li

et al., 2012). Sequence homologs of AGG3 are present in

gymnosperms and angiosperms but not in other organisms

(Trusov et al., 2012). Analysis of the recently available Camelina

sequence database (Liang et al., 2013) revealed the existence of a

protein sequence that shows extremely high homology with

AGG3 (Figure 1a). The protein, tentatively named type III CsGc, iswidely expressed in Camelina plants with highest expression in

seeds and mature leaves (Figure 1b).

To evaluate the role of AGG3 in conferring increased biomass

and seed production, transgenic Camelina plants were generated

using two types of constructs: CaMV35S:AGG3 expressing AGG3

cDNA with a constitutive CaMV35S promoter and Glycinin:AGG3

expressing AGG3 cDNA with a seed-specific, soybean glycinin

promoter (Figure 1c). The constructs also included a DsRed

reporter gene for visual selection of transgenic seeds and a Bar

gene for basta resistance in transgenic plants. Camelina plants

were transformed using a modified floral-dip method (Lu and

Kang, 2008), and multiple independent overexpression lines that

exhibited a 3 : 1 segregation ratio in T2 generation were isolated,

selfed and grown to homozygosity. The T3 homozygous lines

were analysed for increased levels of transgene expression by

quantitative real-time PCR (qRT-PCR) in the seedlings of

CaMV35S:AGG3 lines and in the seeds of Glycinin:AGG3 plants

and compared with plants containing respective empty vectors

(EV). Three independent CaMV35S:AGG3 transgenic lines show-

ing ~63-, 219- and 243-fold higher expression levels compared

with the CaMV35S EV (35S:EV) line and three independent

Glycinin:AGG3 lines showing ~350-, 38- and 16-fold higher

expression levels compared with the glycinin EV (Glycinin:EV) line

(Figure 1d) were selected, and the progeny of these seeds were

used for further phenotypic analyses.

Overexpression of AGG3 gene in Camelina leads tohigher biomass production

Camelina is being developed as a model for herbaceous bioen-

ergy crops, and genetic improvement in biomass yield is a major

target trait, in addition to higher oil production (Ghamkhar et al.,

2010; Nguyen et al., 2013). To evaluate the effect of AGG3

overexpression on different biomass-related traits, twenty-four

plants from each transgenic line and from the EV line were grown

side by side, and data were recorded for various growth

parameters every 2–3 days, until the plants reached maturity

(10 weeks). The entire experiment was repeated twice, with

different batches of seeds and at different times of the year.

Overall, a significant increase in vegetative growth starting from

the initial stages of development was observed in the CaMV35S:

AGG3 transgenic lines. The number of leaves was higher in

CaMV35S:AGG3 lines in successive weeks of growth (~18%,

34% and 26% more at the 3rd week and ~12%, 45% and 40%

more at the 5th week in different lines, respectively) (Figure 2a).

Moreover, leaf length, leaf width and internodal distance were

also appreciably enhanced in overexpression lines compared with

the EV lines (Figure S1). The overexpression lines grew faster and

exhibited overall bigger plant height compared with the EV lines

with ~9%, 21% and 33% increase at the 3rd week and ~17%,

35% and 34% increase by the 5th week of growth in three

different lines (Figure 2b, c, S2a). These plants also had signif-

icantly more branches by this time (Figure S2c). The EV plants

eventually reached the same height as the CaMV35S:AGG3

plants (Figure S2b) but had fewer leaves, branches and flowers.

The overexpression lines flowered earlier, with the first flower

appearing 5–8 days ahead of their appearance in the EV lines

(Figure S3a). These data suggest that higher biomass production

in Camelina due to AGG3 overexpression is a result of an overall

faster plant growth.

Overexpression of AGG3 gene in Camelina results inhigher seed yield

Improvements in seed yield and oil content are the key targets for

the biotechnological modification of oilseed crops. Mutations in

ª 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 12, 49–59

Swarup Roy Choudhury et al.50

the AGG3 homologs in rice result in changes in seed size, seed

length and panicle branching (Fan et al., 2009; Huang et al.,

2009; Mao et al., 2010). Similarly, changes in the expression of

the AGG3 gene in Arabidopsis by T-DNA knockout or overex-

pression lead to altered flower and seed sizes (Chakravorty et al.,

2011; Li et al., 2012). However, whether such changes in seed

size have any effect on the overall seed composition, seed viability

or carbon partitioning has not been evaluated. Our data show

that, similar to the effect on vegetative growth, the reproductive

growth of Camelina transgenic lines was also positively affected

by overexpression of AGG3. The CaMV35S:AGG3 plants not only

flowered earlier but had more flowers per plant compared with

the EV lines (Figure S3a, b). The flower size was larger with ~15%increase in sepal and petal length (Figure 3a, S3c, d, e). The

increase in flower size and number was also reflected in the

transgenic fruits that were larger. This increase in size was also

accompanied by a 15%–20% increase in fruit number per plant

(Figure 3a,b).

We evaluated three different traits for seed yield in CaMV35S:

AGG3 and Glycinin:AGG3: seed size, seed mass and seed number

per fruit. The seed size, measured as the area of seeds, was ~20%and 35% higher in different CaMV35S:AGG3 and Glycinin:AGG3

lines, respectively, compared with their corresponding EV controls

(Figure 3c,d). Similar to seed size, seed weight and seed number

were also higher in both CaMV35S:AGG3 and Glycinin:AGG3

lines. Specifically, a 15%–35% increase in seed weight was

observed for different CaMV35S:AGG3 lines, and a 25%–40%increase in seed weight was observed for different Glycinin:AGG3

Figure 1 Overexpression of Arabidopsis AGG3

gene in Camelina. (a) Protein sequence alignment

of AtAGG3 with type III CsGc protein using three

different sections of Camelina transcriptome

representing isogroup11522|isotig11955, lcl|

scaffold821, scaffold4928 (Liang et al., 2013).

Conceptually translated protein sequences from

the transcript were used for the BLAST analysis

against Arabidopsis AGG3 sequence.

(b) Expression of type III CsGc transcript in different

tissues of Camelina. Primary root and hypocotyl

were collected from 4-day-old seedlings, and

additional tissues were collected from 6-week-old

plants. qRT-PCR amplifications were performed

three times independently for each target, and the

data were averaged. The expression values across

different tissue types were normalized against

Camelina Actin gene expression, which was set at

100. Error bars represent the standard error of the

mean (�SE). (c) Generation of constructs for

constitutive (CaMV35S) and seed-specific glycinin

promoter-driven overexpression of AGG3. The

selection markers, Ds-Red and Bar are driven by

CMV promoter and Nos promoter, respectively.

(d) qRT-PCR analyses of AGG3 expression levels in

3-day-old seedlings and seeds of CaMV35S:AGG3

and Glycinin:AGG3 transgenic Camelina lines,

respectively. The expression was normalized to

Actin gene, and data presented are mean values

of three biological replicates. Error bars represent

standard errors (�SE). Expression in empty vector

lines (EV) is set at 1.


Camelina yield enhancement by AGG3 overexpression 51

lines compared with their EV controls (Figure 3e). The seed

number per fruit was only modestly higher in CaMV35S:AGG3

plants but showed ~15% increase in Glycinin:AGG3 (Figure S4).

The increased number, weight and size of seeds in overexpression

lines lead to significantly higher seed yield per plant compared

with the EV lines (Figure 3f).

To ascertain that no adverse effect on the quality of seeds

resulted from the overexpression of AGG3, the seeds from the

transgenic and EV lines were tested for seed moisture content,

seed germination potential and seed viability. No differences were

observed in the seed quality of either CaMV35S:AGG3 or

Glycinin:AGG3 plants (Figure S5a,b).

The transgenic seeds were evaluated for their oil quantity per

seed, per plant and for oil composition. The percentage of oil on

seed mass basis remained unchanged in the transgenic seeds,

suggesting no difference in the carbon partitioning (Figure 4a) or

overall oil composition (Table 1) due to AGG3 overexpression.

However, the higher total seed mass and seed number per plant

resulted in significantly increased overall oil yield. The oil content

of EV lines was ~2.9 mg per 10 seeds, which increased to ~3.3–4 mg per 10 seeds in overexpression lines (Figure 4b). Moreover,

because the overexpression lines also produced more seeds per

plant, a net increase of up to 20%–35% and 25%–55% in oil

content per plant was observed in CaMV35S:AGG3 and Glycinin:

AGG3 lines, respectively, compared with their corresponding EV

controls (Figure 4c). Taken together, these data show that AGG3

overexpression has a substantial effect on seed-related traits and

consequently on seed and oil yield.

Camelina plants overexpressing AGG3 exhibit higherrates of net photosynthesis and higher stomatalconductance

To investigate the physiological basis of higher growth rates and

yield in overexpression lines, we measured the rates of net

photosynthesis and stomatal conductance. The photosynthetic

rate directly affects the accumulation of starch in vegetative

tissues, which is ultimately responsible for higher biomass.

Similarly, starch that accumulates in leaves contributes to the

seed size through translocation of photosynthates, a prerequisite

for increased seed weight and size. The net photosynthesis,

measured from vegetative growth stage plants, using the 4th, 5th

and 6th leaves starting from the apex, was significantly higher in

overexpression lines compared with the EV controls (Figure 5a).

As photosynthetic efficiency is correlated with the rate of

stomatal conductance, a considerably higher rate of stomatal

conductance was also observed in overexpression lines compared

with the EV lines (Figure 5b). This higher stomatal conductance

also resulted in a higher rate of transpiration in AGG3 overex-

pression lines (Figure 5c).

At the cellular level, overexpression of AGG3 seems to affect

cell division as shown by the higher numbers of dividing cells in 3-

day-old growing roots (Figure S6). At later stages of growth,

when the transgenic seedlings are significantly larger than the EV

control lines, no differences were observed in cell sizes of roots,

leaves or flower petals (Figure S7). This suggests that an increase

in cell number is responsible for the larger size of different organs

in transgenic lines, consistent with what has been reported for

AGG3 overexpression in Arabidopsis (Li et al., 2012).

Camelina plants overexpressing AGG3 showhyposensitivity to ABA, sucrose and NaCl in seed-relatedtraits

The AGG3 gene was originally characterized in Arabidopsis for its

role in the regulation of ABA-mediated signalling pathways

(Chakravorty et al., 2011). The agg3 seeds are hypersensitive to

ABA for inhibition of germination and postgermination growth,

and hyposensitive to ABA for the inhibition of stomatal opening,

similar to the phenotypes observed in the Arabidopsis Ga (gpa1)

and Gb (agb1) mutants (Chakravorty et al., 2011; Fan et al.,

2008; Pandey et al., 2006; Wang et al., 2001). The effect of ABA

on Arabidopsis plants overexpressing AGG3 has not been

evaluated. Similarly, whether rice GS3 or DEP1 mutants have

differential sensitivities to ABA is not known. Because stress

responses of plants are a critical determinant of yield, we

investigated whether overexpression of AGG3 in Camelina

resulted in altered responsiveness to different stresses. Under

control conditions, seed germination potential of CaMV35S:

AGG3 was comparable to that of the EV controls (Figure S5a).

However, in the presence of ABA, the CaMV35S:AGG3 seeds

exhibited better germination compared with the EV seeds. In the

presence of 3 and 5 lM ABA, only 70% and 45% EV seeds

germinated, respectively, compared with ~80%–90% and 55%–80% germination seen in different CaMV35S:AGG3 lines at 24 h

(a)

(b)

(c)

Figure 2 Vegetative growth parameters of transgenic Camelina plants.

(a) Number of leaves in CaMV35S:AGG3 overexpression and EV lines at

3- and 5-week-old plants. L1, L2 and L3 represent three independent

overexpression lines. (b) Height of plants from the EV and overexpression

plants (3 and 5 weeks). Data presented are mean values of 24 plants from

each line, and error bars represent standard errors (�SE). Significant

difference at *P < 0.05 and **P < 0.005, respectively (Student’s t-test).

(c) Representative picture of 5-week-old plants from EV and overexpression

lines. Additional growth parameters are listed in Figures S1 and S2.



(Figure 6a,b). This response was relatively more pronounced in

the growth media without sucrose compared with the growth

media containing 1% sucrose (Figure 6a,b). The trend continued

in the later time points where the CaMV35S:AGG3 overexpres-

sion lines consistently showed better germination (Figure S8).

Similar results were observed for the Glycinin:AGG3 seeds (Figure

S9, S10). The CaMV35S:AGG3 plants also showed less sensitivity

to ABA for primary root length inhibition and lateral root number

(Figure 6b,c), whereas no significant differences were observed

between the EV and CaMV35S:AGG3 plants on standard 1%

sucrose containing 0.5X MS media at this stage of growth. The

root growth of CaMV35S:AGG3 was also less sensitive to osmotic

stress induced due to the presence of 0.4 M Sucrose (Figure S11).

In addition, CaMV35S:AGG3 transgenic seedlings showed

hyposensitivity to salt stress in the presence of 100 mM NaCl

(Figure S11), suggesting a general improvement of stress

tolerance in the CaMV35S:AGG3 plants.

AGG3 overexpression results in ABA hypersensitivestomatal responses and better stress tolerance intransgenic Camelina plants

G-proteins regulate ABA sensitivities in a tissue-dependent

manner; the knockout mutants of Arabidopsis Ga, Gb and Gc3are hypersensitive to ABA for germination and postgermination

growth but hyposensitive to stomatal responses (Chakravorty

et al., 2011; Fan et al., 2008; Pandey et al., 2006; Wang et al.,

2001). This tissue-specific regulation of ABA responsiveness was

also evident in transgenic Camelina plants overexpressing AGG3.

While the germination and early seedling growth of overexpres-

sion lines exhibit hyposensitivity to ABA (Figure 6), hypersensitiv-

ity to ABA was observed during regulation of stomatal responses.

Treatment with 25 lM ABA led to modest differences (~10%) in

stomatal aperture of EV plants, whereas the stomata of overex-

pression lines displayed significantly smaller apertures, with up to

(a)

(c)

(d)

(e)

(f)

(b)

Figure 3 Flower-, fruit- and seed-related

phenotypes of Camelina plants overexpressing

AGG3. (a) Increased sizes of mature flowers and

mature fruits, and (b) increased fruit number per

plant in CaMV35S:AGG3 Camelina plants. L1, L2

and L3 represent three independent transgenic

lines. Seed-specific traits were evaluated in both

CaMV35S:AGG3 and Glycinin:AGG3 lines and

compared with their respective EV controls.

(c) Comparison of seed sizes in CaMV35S:AGG3

(35S) and Glycinin:AGG3 (Glycinin) overexpression

lines with the EV lines. (d) Seed area (mm2 per 50

seeds), (e) seed weight (g per 100 seeds) and

(f) total seed weight per plant were measured.

Data presented are mean of 24 plants from each

line, and error bars represent standard errors

(�SE). For seed weight and seed area, three

replicates were taken from each plant. Significant

difference at *P < 0.05 and **P < 0.005,

respectively (Student’s t-test).



30% reduction in pore size (Figure 7a,b). The hypersensitivity of

stomata to ABA was also verified in detached-leaf water loss

experiments where CaMV35S:AGG3 exhibited lower rate of

water loss compared with the EV plants as measured by the fresh

weight loss of detached leaves from 1 to 8 h (Figure 7c). While

the CaMV35S:AGG3 plants lost ~55% water during an 8-h

period, ~75% water loss was observed in EV plants.

We further explored the role of AGG3 in providing drought

tolerance. Because Camelina is inherently relatively drought

tolerant, a large effect of low water stress was not obvious.

However, when 10-day-old plants were grown without water for

an additional 10 days, followed by re-watering, and drought

recovery was estimated by evaluating the number of surviving

plants after 7 days, differences were observed between the EV

and overexpression lines. In five independent experiments, less

than 40% of EV plants survived this drought/recovery regime,

whereas the survival of different CaMV35S:AGG3 lines varied

from 50% to 60% (Figure 7d). The Glycinin:AGG3 transgenic

lines showed no difference in survival from the EV lines as

expected (Figure 7d).

Discussion

The role of AGG3 in yield enhancement in Camelina

With the world population expected to reach 9 billion by 2050,

ever-rising demand for food, feed, fibre and fuel cannot be

overemphasized. To satisfy this demand, crop yield improve-

ment has been one of the major goals of plant biology

research. Based on the extensive studies in model plants over

the years, multiple genes regulating a variety of different

pathways have been suggested to improve yield and/or provide

stress tolerance. However, barring few exceptions, the transla-

tion of such knowledge to important food and fuel crops is

only beginning to be evaluated (Parry and Hawkesford, 2010,

2012; Peterhansel and Offermann, 2012; Rojas et al., 2010;

Ruan et al., 2012).

(a)

(b)

(c)

Figure 4 Oil content in transgenic Camelina

plants. (a) FAME was extracted from EV and

overexpression Camelina seeds and analysed

using gas chromatography. (a) Seed oil content

(percentage of oil/seed) and (b) mass of oil/10

seeds were measured in different overexpression

lines and compared with their respective EV

control. Six biological replicates were used, and

data were averaged. (c) Mass of oil/plant was

calculated from total seed weight in CaMV35S:

AGG3 and Glycinin:AGG3 overexpression lines

compared with EV lines. Data presented are mean

value of 6 plants of each line, and error bars

represent standard error (�SE). Significant

difference at *P < 0.05 and **P < 0.005,

respectively (Student’s t-test).

Table 1 Fatty acid composition of Camelina seed oils in different CaMV35S:AGG3 and Glycinin:AGG3 overexpression and empty vector (EV)

lines. Data represent mean values of 6 individual plants.

Fatty Acid 16:00 18:00 18:01 18:02 18:03 20:00 20:01 20:02 20:03 22:00 22:01

CaMV35S:AGG3

EV 7.63 2.88 11.49 23.19 33.03 2.06 12.76 2.22 1.23 0.39 3.07

L1 7.58 2.95 11.84 23.34 32.33 2.15 12.87 2.19 1.18 0.41 3.11

L2 7.06 2.78 10.12 20.16 37.19 2.40 12.64 2.21 1.47 0.4 3.45

L3 7.35 2.67 10.73 22.66 36.18 2.09 11.64 2.17 1.23 0.39 2.83

Glycinin:AGG3

EV 7.14 2.59 11.09 21.57 34.32 2.08 13.60 2.39 1.37 0.40 3.40

L1 7.23 3.15 10.82 21.06 34.86 2.55 12.89 2.11 1.33 0.49 3.46

L2 7.44 3.21 12.59 22.49 32.60 2.30 12.75 2.03 1.15 0.42 2.96

L3 7.59 3.00 12.16 23.46 32.12 2.15 12.69 2.16 1.13 0.40 3.09



Type III Gc proteins have been proposed to be major regulators

of yield-related traits such as seed size, seed number, panicle

branching and abiotic stress tolerance, based on the studies in

Arabidopsis, rice and soybean (Chakravorty et al., 2011; Fan

et al., 2009; Huang et al., 2009; Li et al., 2012; Roy Choudhury

and Pandey, 2013). While the Arabidopsis data are relatively

straightforward, the markedly small size of Arabidopsis seeds and

relatively modest phenotypes necessitate their further evaluation.

The rice data, on the other hand, are complex. Specific mutations

that allow for the expression of different truncated versions of the

same protein lead to distinct, sometimes contrasting phenotypes

(Botella, 2012; Lu and Kang, 2008; Mao et al., 2010). Therefore,

further studies are required to establish the potential positive

effects of type III Gc proteins and to expand their scope on

agronomically important plants. We chose to investigate the

potential of the AGG3 gene in Camelina, because it is an

emerging biofuel crop, genetically tractable and is closely related

to Arabidopsis. Importantly, its larger plant stature and seed size

facilitate detailed quantitative evaluation of various biomass and

seed-associated traits. A sequence homolog of AGG3 gene was

identified in the Camelina genome, and the endogenous gene

exhibited widespread expression (Figure 1a,b). To minimize any

potential deleterious effects of high-level constitutive expression

of AGG3 gene from a constitutive promoter (CaMV35S), trans-

genic plants were also generated with a seed-specific (glycinin)

promoter (Figure 1c).

Overexpression of AGG3 with CaMV35S promoter led to

higher overall biomass production as determined by an increased

number of leaves and number of branches per plant. During the

vegetative growth phase, the CaMV35S:AGG3 plants grew

significantly faster than the control plants (Figure 2, S1, S2). This

faster growth was accompanied by higher rates of photosynthesis

and stomatal conductance as well as more cell division (Figure 5,

S6, S7). However, with transition to the reproductive phase,

resources were mostly mobilized towards the production of

higher numbers of flowers and fruits (Figure 3). As a result, after

10 weeks of growth, the overall height of the CaMV35S:AGG3

plants was not significantly different from the EV plants (Figure

S2b), but they had a substantially higher number of leaves,

branches, fruits and seeds per plant (Figure S2c, Figure 3). An

increase in seed size, weight and seed number per plant

altogether resulted in a significantly higher yield in transgenic

plants compared with EV plants (Figure 3f). Furthermore, no

differences were observed in seed quality between the control

and overexpression plants when comparing the seed germination

potential, viability or carbon partitioning (Figure 4, S5a,b).

One of the main goals of this study was to estimate the effect

of AGG3 overexpression on oil production. Camelina seeds

accumulate 25%–40% oil per seed mass basis depending on

specific growth conditions (Nguyen et al., 2013). We consistently

recovered ~30% oil in WT and EV Camelina seeds. In the

overexpression lines, the percentage of oil per seed did not

change; however, larger seed sizes and more seeds per plant

resulted in significantly more oil production per plant (Figure 4).

No major changes were observed in lipid composition (Table 1),

suggesting that there was no effect on the oil quality between

control and transgenic plants. The seed-specific traits were similar

in both CaMV35S:AGG3 and Glycinin:AGG3, suggesting that a

seed-specific promoter can be used in situations where improved

vegetative growth may not be desired. It should be noted that

while there is a need to improve the quality of oil in Camelina to

make it more usable for biofuel application (Nguyen et al., 2013),

the demonstration that manipulation of fundamental develop-

mental and physiological processes can lead to higher oil yield is

significant. Combining different approaches, geared towards

improving quality as well as the quantity of seed oil, is therefore

likely to result in higher amounts of desirable oil types in oilseed

plants.

The role of AGG3 in regulating abiotic stress responses

Engineering stress tolerance is an important aspect of overall

plant productivity (Carmo-Silva and Salvucci, 2012; Parry and

Hawkesford, 2012; Rojas et al., 2010). Interestingly, AGG3 gene

in Arabidopsis was initially identified as the missing piece of the

G-protein heterotrimer that regulates ABA signalling in conjunc-

tion with the Ga and Gb proteins (Chakravorty et al., 2011). The

previously identified Gc proteins of Arabidopsis, AGG1 and

AGG2, are not involved in the regulation of ABA signalling but

mediate biotic stress responses (Chakravorty et al., 2011; Thung

et al., 2012; Trusov et al., 2008).

Multiple abiotic stresses affect ABA signalling in plants, and

many different signalling modules are involved in the regulation

(a)

(b)

(c)

Figure 5 Rate of photosynthesis, stomatal conductance and

transpiration of CaMV35S:AGG3 Camelina plants. (a) Net photosynthesis,

measured as the amount of CO2 assimilated per second, (b) net stomatal

conductance, measured as the amount of H2O transferred per second,

and (c) net transpiration rate, measured as the amount of water loss per

second, were determined on individual 4th, 5th and 6th leaves (from

apical bud) of 4-week-old empty vector (EV) and CaMV35S:AGG3

overexpression lines using a Li-COR 6400 gas exchange system. Six

biological replicates of each line and five measurements for each leaf were

used for data analysis. Error bars represent standard errors (�SE) and

significant difference at *P < 0.05 (Student’s t-test).



of ABA-mediated responses. Abscisic acid inhibits seed germina-

tion and primary root growth and promotes stomatal closure to

secure water loss. Attempts to modify ABA-related processes for

biotechnological improvements have met unique challenges

(Nakashima and Yamaguchi-Shinozaki, 2013; Qin et al., 2011).

If ABA hyposensitivity is engineered in whole plants, the seeds

show better germination, but the plants are relatively more

sensitive to drought. In contrast, when engineering ABA hyper-

sensitivity in whole plants, the drought responses are improved

but seed germination is adversely affected (Wang et al., 2005,

2009). The use of tissue-specific promoters has helped address

some of these challenges (Jammes et al., 2009; Oh et al., 2011;

(a) (b)

(c) (d)

(a) (b)

(c) (d)

Figure 6 Effect of ABA on seed germination and primary root length of CaMV35S:AGG3 Camelina plants. Germination of three different overexpression

lines compared with EV control lines at 24-h time point (a) in the absence and (b) in the presence of 1% sucrose. All seeds germinated on control plates

(without ABA) at this time point. Data recorded at additional time points are shown in Figure S8. The experiment was repeated three times, and data were

averaged, n = 60 per line for each experiment. (c) Seeds were germinated on 0.5X MS for 24 h followed by transfer to plates containing 0, 30 or 50 lM

ABA. Primary root length was measured after 3 days of vertical growth, n = 30. (d) Seeds were germinated on 0.5X MS for 24 h followed by transfer to 0,

10 or 20 lM ABA. Lateral root number/primary root was calculated from transgenic lines after 5 days of vertical growth, n = 30. Root growth experiments

were repeated three times, and data were averaged. Error bars represent standard errors (�SE) and significant difference at *P < 0.05 (Student’s t-test).

Figure 7 ABA-induced stomatal aperture changes and rate of water loss from transgenic Camelina plants. (a) Promotion of stomatal closure and

(b) inhibition of stomatal opening in EV and three different CaMV35S:AGG3 plants in response to 25 lM ABA. Data are mean of 3 biological replicates.

Approximately 100 stomatal apertures from each line were measured for each replicate. (c) Rate of water loss from detached leaves of 2-week-old plants

was measured as percentage of initial fresh weight after the indicated time periods. The plants were maintained at 60% relative humidity. Values are mean

�SE of five individual plants per transgenic lines. The entire experiment was replicated three times. Data were compared using the Student’s t-test at the

95% significance level. (d) Watering of 10-day-old plants was stopped for the next 10 days and then rewatered for 7 days. Number of survived plants/total

plants was counted in five independent experiments. Error bars for all experiments represent standard errors (�SE) and significant difference at *P < 0.05

(Student’s t-test).



Wang et al., 2009), but such promoters may not always be easily

accessible or may not work efficiently in all plants. Proteins that

inherently regulate signalling in a tissue-specific manner (e.g. G-

proteins) therefore provide a viable alternative to these challenges

as a constitutive promoter may be used to achieve desirable

response. G-proteins are positive regulators of ABA-dependent

stomatal responses but negative regulators of ABA-dependent

germination and postgermination growth (Fan et al., 2008;

Pandey et al., 2006; Wang et al., 2001). The data presented in

Figure 6 and 7 substantiate the tissue-specific regulation of ABA

signalling by overexpression of AGG3 in Camelina. The transgenic

seeds displayed less sensitivity to ABA, osmotic stress and salt

stress during germination and postgermination growth (Fig-

ures 6, S8, S9, S10, S11). In addition, the stomata of CaMV35S:

AGG3 plants were more sensitive to ABA, and a relatively better

drought recovery was observed in these plants compared with EV

control or Glycinin:AGG3 plants (Figure 7). It should be noted

that under nonstressed conditions, the stomatal conductance and

rate of transpiration in CaMV35S:AGG3 plants were higher than

in EV plants; however, higher ABA sensitivity of stomata in these

plants possibly leads to faster stomatal closure under stress

conditions and results in less water loss and better drought

recovery. Taken together, these results suggest a clear, positive

role for AGG3 overexpression on multiple growth and develop-

ment pathways as well as stress response, which leads to a

significant increase in productivity.

As per the established signalling mechanisms, Gc proteins

always act as obligate dimers with Gb proteins. While the exact

number of subunits of each G-protein remains to be identified in

Camelina, it is conceivable that additionalGcproteins are present inthe Camelina genome based on the subunit diversity and its

relationship to plant ploidy (Bisht et al., 2011; Roy Choudhury

et al., 2011; Trusov et al., 2012). It is therefore possible that by

overexpressing the Gc subunit alone, the quantity of Gb protein

becomes limited and/or the stoichiometry between different Gbccombinations is affected. Future research, aimed towards delin-

eating the mechanism of G-protein signalling during control of

these important agronomic traits, is required to evaluate the roles

of specific G-protein subunits and their combinations. However,

the data presented here do emphasize that by modifying funda-

mental G-protein-regulated physiological processes, such as sto-

matal conductance and cell numbers, significant biotechnological

traits can be engineered in agronomically important plants.

Experimental procedures

Plant growth conditions and morphological analyses

Camelina (Camelina sativa, variety Suneson) seeds were sterilized

in 70% ethanol, 30% bleach and 0.1% Triton-100 X for 30 min

followed by extensive washing with sterile water and transferred

on 0.5X MS (pH 5.7), 1% agar, 1% sucrose medium. After

stratification at 4 °C for 48 h, seeds were germinated at 16-h

light, 8-h dark, 23 °C regime. After 4 days, seedlings were

transferred to soil-rite (Fafard 3B mix) and grown in the

greenhouse. At maturity (12–14 weeks), seeds were harvested,

counted, weighed and photographed. Seed area was measured

using IMAGE J (http://rsb.info.nih.gov/ij/). Plant height, leaf

number, leaf size, branch number, internodal distance, flowering

time, flower and fruit number and morphology of control and

transgenic Camelina plants were recorded for the entire lifecycle

at 2–3 day intervals. Seed viability was tested by soaking seeds in

2,3,5 triphenyltetrazolium chloride solution (1% w/v) in darkness

at 30 °C for 48 h (Wharton, 1955). Formazan production in

viable seeds containing active dehydrogenases led to strong red

coloration. Heat-killed seeds (incubated at 100 °C for 1 h) were

used as negative control.

Seed germination assays were performed according to

Pandey et al. (2006) in 0.5X MS, 1% agar medium in the

presence of different concentrations of ABA with or without

1% sucrose. Seeds of each genotype were plated on 150-mm

plates and stratified in darkness at 4 °C for 48 h. Plates were

transferred to growth chambers at 16-h light/8-h dark, 23 °Cregime. Germination was recorded starting 12 h up to 60 h and

expressed as a percentage of total seeds that showed an

obvious protrusion of radicle. For evaluating the effect of ABA

on root growth, seeds were first germinated and grown on

0.5X MS, 1% agar, 1% sucrose for 24 h followed by transfer

to treatment plates containing different concentrations of ABA

and grown for additional 3–5 days. For each experiment, empty

vector lines were grown side by side on the same plates. Each

plate had four seedlings per genotype, and eight plates were

used for each assay. For evaluating the effect of high sucrose,

seeds from EV and CaMV35S:AGG3 lines were germinated on

0.5X MS in the presence of either 1% (control) or 0.4 M

(13.7%) sucrose. Primary root length was measured from

transgenic lines after 4 days of vertical growth. To evaluate the

effect of high salt, EV and CaMV35S:AGG3 seeds were

germinated on 0.5X MS media without sucrose or with 1%

sucrose and in the presence of 100 mM NaCl. To assess

whether MS salt concentration had any effect on NaCl

sensitivity, the experiment was also repeated at 0.1X, 0.25X

and 0.5X strength MS salt concentrations. Primary root length

was measured after 5 days of growth.

To measure cell size, roots from 3-day-old plants grown on

0.5X MS media were cut near the actively growing tip region and

stained with 1X propidium iodide for 15 min followed by

destaining with water. The roots were imaged at 20X magnifi-

cation with the Zeiss LSM510 laser scanning confocal microscope

(Carl Zeiss, Thornwood, NY). To measure cell sizes of mature

plant parts, epidermal layers of leaves and flower petals were

visualized with light microscopy at 20X magnification, and cell

sizes were quantified using IMAGE J (http://rsb.info.nih.gov/ij/).

RNA isolation and qRT-PCR analysis

RNA was isolated from Arabidopsis and Camelina tissues using

TRIzol� RNA (Invitrogen, Carlsbad, CA) and first-strand cDNA was

prepared by SuperScript� III First-Strand Synthesis System (Invi-

trogen). qRT-PCR were performed as described previously (Bisht

et al., 2011). The oligonucleotides used for PCR are listed in

Supplemental Table S1.

Generation of transgenic plants

Full-length AtAGG3 (At5 g20635) was amplified using Platinum�

Pfx (Invitrogen) from Arabidopsis flower cDNA and confirmed by

sequencing. The seed-specific overexpression construct was gen-

erated by the insertion ofAGG3 cDNA into amodified pBinGlyRed1

vector between glycinin promoter and terminator at EcoRI andNruI

sites. The constitutive overexpression construct was generated by

replacing the glycinin promoter of pBinGlyRed1 vector with

CaMV35S promoter at BamHI and EcoRI sites. The overexpression

constructs and empty vectors were introduced intoAgrobacterium

tumaefaciens strain GV301 by electroporation.

Six-week-old wild-type Camelina plants were transformed

using floral dip (Lu and Kang, 2008). Transgenic seeds (T1) were



visually selected by Ds-Red expression and transferred to soil for

growth to maturity. Seeds from lines displaying a 3 : 1 segrega-

tion of T2 seeds on the basis of Ds-Red signal were selected for

next generation. Homozygous T3 seeds of the transgenic plants

were selected, and three independent transgenic lines exhibiting

maximum expression of AtAGG3 gene were selfed and used for

further analyses.

Seed oil content measurement

Fatty acid methyl esters (FAME) were prepared from mature

Camelina seeds according to Lu et al. (2013). Tri-17 : 0 triacyl-

glycerol was included as an internal standard. FAME analysis were

performed by gas chromatography (Trace GC; ThermoQuest, East

Lyme, CT) on a HP-INNOWAX (Agilent technologies, Santa Clara,

CA) column (30 m 9 0.25 mm i.d., 0.25-lm film thickness)

using helium gas, equipped with a flame ionization detector (AI/

AS 3000) injector. Methyl esters was identified by comparison of

reaction times of standard FAME, and a normalization technique

was used for quantitation with CHROMQUEST 5.0, version 3.2.1

(Thermofisher Scientific, Waltham, MA).

Measurement of photosynthetic rate and stomatalconductance

Parameters of leaf photosynthesis rate including CO2 assimilation,

stomatal conductance and transpiration rate were measured with

a portable photosynthetic system, LI6400XT (Li-COR, Lincoln, NE).

The conditions in the leaf chamber were calibrated similar to

those in the greenhouse where plants were growing: 500 lmol/

m2/s photosynthetic photo flux density, 400 lmol/mol CO2,

23 °C and 60% relative humidity. Measurements were taken on

the 4th, 5th and 6th open leaves from the apical bud.

Stomatal aperture and water loss assays

For ABA-induced promotion of closure measurement, fully

opened leaves of 2-week-old plants were floated, abaxial side

down, in a solution of 20 mM KCl, 1 mM CaCl2 and 5 mM MES-

KOH (pH 6.15) for 3 h under cool white light (200 lmol/m2/s) at

23 °C to induce stomatal opening. ABA (25 lM) was added and

the leaves were incubated for additional 3 h under light to

promote closure. For inhibition of stomatal opening, the leaves

were floated in 10 mM KCl, 7.5 mM potassium iminodiacetate

and 10 mM MES-KOH (pH 6.15) in darkness for 3 h to ensure

complete stomatal closure. The leaves were transferred to light

(200 lmol/m2/s) in the presence of 25 lM ABA for additional 3 h.

For both sets of experiments, equimolar amount of EtOH was

used as control for non-ABA treated leaves. Epidermal strips were

isolated from the leaves at the end of incubation and wet-

mounted on microscopic slides. Images were recorded using a

wide-field microscope fitted with digital camera and analysed

using IMAGE J. The experiments were performed double blind.

For water loss measurement, detached leaves from 2-week-old

plants were exposed to cool white light (125 lmol/m2/s) at 23 °Cand 60% relative humidity. Leaves were weighed at indicated

time intervals, and the loss of fresh weight (%) was used to

estimate water loss.

Whole-plant drought tolerance estimation

Camelina plants were grown in the greenhouse in a block

arrangement. Each block contained 2 EV and 2 plants from three

different overexpression lines. Six independent blocks were used

for each experiment. The position of plants was varied in each

block. Ten-day-old, well-watered plants were used for drought

experiments. The plants were grown without water for additional

10 days, followed by re-watering for 7 days. Drought tolerance

was determined by quantifying the number of surviving plants/

total number of plants.

Acknowledgements

We thank Dr. Jan Jaworski, Jia Li and Matthew Shipp for their

help with various Camelina-related resources, Dr. Noah Fahlgren

for assistance with retrieving Camelina sequences and Christine E.

Barnickol for editing of the manuscript. This work was supported

by the Agriculture and Food Research Initiative (Grant no. 2010–65116–20454) to SP. AJR was supported by a NSF-REU grant

(NSF-DBI-REU-1156581).

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Supporting information

Additional Supporting information may be found in the online

version of this article:

Figure S1 Leaf phenotype and internodal distance of transgenic

Camelina plants.

Figure S2 Vegetative growth parameters of CaMV35:AGG3

overexpression lines.

Figure S3 Flower-related growth parameters of CaMV35:AGG3


Figure S4 Seed number per fruit in overexpression lines.

Figure S5 Seedviability-relatedtraitsoftransgenicCamelinaplants.

FigureS6 Increasedcelldivision inCaMV:AGG3 lines.

FigureS7Cell sizesofEVandoverexpression lies.

Figure S8 ABA hyposensitive seed germination of CaMV:AGG3


Figure S9 Seedgermination ofGlycinin:AGG3 overexpression lines

areABAhyposensitive inabsenceofsucrose.

Figure S10ABA hyposensitive seed germination ofGlycinin:AGG3


Figure S11 High sucrose and NaCl hyposensitivity of CaMV:AGG3

plants.

Table S1 Primer sequences used in experiment.



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