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... status of Ga protein. GDP-Gabc trimeric complex represents the inactive state ... Arabidopsis thaliana has only one Ga, one Gb and three Gc- proteins (Temple .... reporter gene for visual selection of transgenic seeds and a Bar gene for basta ...

Plant Biotechnology Journal (2014) 12, pp. 49–59

doi: 10.1111/pbi.12115

Constitutive or seed-specific overexpression of Arabidopsis G-protein c subunit 3 (AGG3) results in increased seed and oil production and improved stress tolerance in Camelina sativa Swarup 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.

Summary Heterotrimeric 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, Gb and Gc subunits, switches between active and inactive conformation depending on the guanine nucleotide-bound status of Ga protein. GDP-Gabc trimeric complex represents the inactive state of signalling. A signal-dependent exchange of GDP for GTP on Ga results 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 Gcproteins (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 discovered 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 Gc proteins, 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 Arabidopsis 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

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50 Swarup Roy Choudhury et al. 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 b subunits 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 b subunits 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). Therefore, further experimental evidence in additional plant systems is required to establish the roles of G-proteins as universal regulators 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 seedrelated 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 overexpressing Arabidopsis 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, is widely 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 showing ~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 to higher biomass production Camelina is being developed as a model for herbaceous bioenergy 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 significantly 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 in higher 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

Camelina yield enhancement by AGG3 overexpression 51

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.

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 overexpression 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

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52 Swarup Roy Choudhury et al. (a)

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.

(b)

Camelina plants overexpressing AGG3 exhibit higher rates of net photosynthesis and higher stomatal conductance

(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.

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,

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 overexpression 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 3day-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 show hyposensitivity to ABA, sucrose and NaCl in seed-related traits 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 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 12, 49–59

Camelina yield enhancement by AGG3 overexpression 53 (a)

(b)

(c)

(d)

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).

(e)

(f)

(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 overexpression 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 hypersensitive stomatal responses and better stress tolerance in transgenic Camelina plants G-proteins regulate ABA sensitivities in a tissue-dependent manner; the knockout mutants of Arabidopsis Ga, Gb and Gc3 are 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 overexpression lines exhibit hyposensitivity to ABA (Figure 6), hypersensitivity 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 overexpression lines displayed significantly smaller apertures, with up to

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

54 Swarup Roy Choudhury et al. (a)

(b) 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).

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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

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

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

CaMV35S:AGG3

Glycinin:AGG3

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

Camelina yield enhancement by AGG3 overexpression 55 (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).

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), transgenic 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 developmental 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 conjunction 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

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56 Swarup Roy Choudhury et al. (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).

of ABA-mediated responses. Abscisic acid inhibits seed germination 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 hypersensitivity 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;

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Camelina yield enhancement by AGG3 overexpression 57 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. Gproteins) 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 (Figures 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 development 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 additional Gc proteins are present in the 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 Gbc combinations is affected. Future research, aimed towards delineating 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 fundamental G-protein-regulated physiological processes, such as stomatal 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 °C regime. 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 magnification 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 (Invitrogen). 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 generated by the insertion of AGG3 cDNA into a modified pBinGlyRed1 vector between glycinin promoter and terminator at EcoRI and NruI 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 into Agrobacterium 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

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58 Swarup Roy Choudhury et al. visually selected by Ds-Red expression and transferred to soil for growth to maturity. Seeds from lines displaying a 3 : 1 segregation 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 triacylglycerol 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 stomatal conductance 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 MESKOH (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 wetmounted 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 °C and 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 overexpression lines. Figure S4 Seed number per fruit in overexpression lines. Figure S5 Seedviability-relatedtraitsoftransgenicCamelinaplants. Figure S6 Increased cell division in CaMV:AGG3 lines. Figure S7 Cell sizes of EV and overexpression lies. Figure S8 ABA hyposensitive seed germination of CaMV:AGG3 overexpression lines. Figure S9 Seed germination of Glycinin:AGG3 overexpression lines are ABA hyposensitive in absence of sucrose. Figure S10 ABA hyposensitive seed germination of Glycinin:AGG3 overexpression lines. Figure S11 High sucrose and NaCl hyposensitivity of CaMV:AGG3 plants. Table S1 Primer sequences used in experiment.

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