Soybean seeds overexpressing asparaginase ... - Wiley Online Library

Physiologia Plantarum 155: 126–137. 2015

© 2015 Scandinavian Plant Physiology Society, ISSN 0031-9317

Soybean seeds overexpressing asparaginase exhibit reduced nitrogen concentration Sudhakar Pandurangana,b , Agnieszka Pajakb , Tara Rintoulb , Ronald Beyaertb , Cinta Hernández-Sebastiàb , Daniel C. W. Brownb and Frédéric Marsolaisa,b,* a Department b Genomics

of Biology, University of Western Ontario, London, Ontario N6A 5B7, Canada and Biotechnology, Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario N5V 4T3,

Canada

Correspondence *Corresponding author, e-mail: [email protected] Received 28 January 2015; revised 18 March 2015 doi:10.1111/ppl.12341

In soybean seed, a correlation has been observed between the concentration of free asparagine at mid-maturation and protein concentration at maturity. In this study, a Phaseolus vulgaris K+ -dependent asparaginase cDNA, PvAspG2, was expressed in transgenic soybean under the control of the embryo specific promoter of the 𝛽-subunit of 𝛽-conglycinin. Three lines were isolated having high expression of the transgene at the transcript, protein and enzyme activity levels at mid-maturation, with a 20- to 40-fold higher asparaginase activity in embryo than a control line expressing 𝛽-glucuronidase. Increased asparaginase activity was associated with a reduction in free asparagine levels as a percentage of total free amino acids, by 11–18%, and an increase in free aspartic acid levels, by 25–60%. Two of the lines had reduced nitrogen concentration in mature seed as determined by nitrogen analysis, by 9–13%. Their levels of extractible globulins were reduced by 11–30%. This was accompanied by an increase in oil concentration, by 5–8%. The lack of change in nitrogen concentration in the third transgenic line was correlated with an increase in free glutamic acid levels by approximately 40% at mid-maturation.

Introduction In soybean [Glycine max (L.) Merr.], seed protein concentration is an important trait for end use and a determinant of crop value. A large number of quantitative trait loci (QTL) and genome-wide association study loci determining population variation in protein concentration have been described (Zhao-Ming et al. 2011, Pathan et al. 2013, Yesudas et al. 2013, Hwang et al. 2014, Qi et al. 2014, Wang et al. 2014, Sonah et al. 2015). However, the corresponding genes remain to be identified (Bolon et al. 2010, Joshi et al. 2013, Lestari et al. 2013). Understanding the factors regulating protein and oil concentration is required to develop high-yielding

soybean varieties, while preserving seed quality (Ainsworth et al. 2012). In general, a negative relationship has been observed between protein and oil concentration, and protein concentration and yield (Brim and Burton 1979), but in some populations, the negative relationship between protein concentration and yield has not been observed (Cober and Voldeng 2000). More recently, a QTL has been reported that is positively associated with both protein and oil concentration (Eskandari et al. 2013a), and polymorphisms in triacylglycerol biosynthetic genes have been associated with variation in oil concentration or yield, without affecting protein concentration (Eskandari et al. 2013b).

Abbreviations – GABA, 𝛾-aminobutyric acid; GUS, 𝛽-glucuronidase; IFS1, isoflavone synthase 1; MU, 4-methylumbelliferone; PCR, polymerase chain reaction; PvAspG1, Phaseolus vulgaris asparaginase 1; QTL, quantitative trait locus; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

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Seed protein accumulation requires the translocation of nitrogenous assimilates from the mother plant to the developing embryo. Assimilates are released from the seed coat, a maternal tissue, into the apoplast, and taken up by the embryo through active transport (Tegeder and Rentsch 2010). In soybean, the amide amino acid asparagine is one the main compounds used for nitrogen translocation in the xylem and in phloem irrigating the developing seed (Matsumoto et al. 1977, McClure and Israel 1979, Rainbird et al. 1984, Hernández-Sebastià et al. 2005). Asparagine is predominant in the embryo at mid-maturation, accounting for approximately 50% of total free amino acids (Rainbird et al. 1984, Hernández-Sebastià et al. 2005). A correlation has been reported between the levels of free asparagine in the developing embryo at mid-maturation and protein concentration in mature seed (Hernández-Sebastià et al. 2005). This correlation has been confirmed in a population of recombinant inbred lines derived from high and low protein parents (Pandurangan et al. 2012). These results are consistent with the positive correlation between transcript levels of asparagine synthetase 1 in source leaves and seed protein concentration (Wan et al. 2006). Asparaginase [EC 3.5.1.1] is the main asparagine catabolic enzyme associated with sink tissues, including developing seeds (Grant and Bevan 1994). There are two subfamilies of asparaginases in higher plants, differing in activation by potassium (Bruneau et al. 2006, Michalska and Jaskolski 2006). K+ -dependent asparaginases have higher catalytic activity than the K+ -independent enzymes. Crystallographic studies have revealed that binding of K+ to the activation loop of recombinant Phaseolus vulgaris asparaginase 1 (PvAspG1) changes its conformation, turning a catalytic switch ON, while binding of Na+ to the same site sets it OFF (Bejger et al. 2014). In Arabidopsis, asparaginases appeared dispensable for seed development (Ivanov et al. 2012). Lack of asparaginase activity in a transfer-DNA (T-DNA) insertion mutant was associated with a doubling of free asparagine in mature seed under low illumination, when a high ratio of nitrogen to carbon is translocated to the seed. The relatively weak impact of asparaginase deficiency in Arabidopsis may also be due, at least in part, to the minor contribution of asparagine as a nitrogen translocation form in the phloem of species from the Brassicaceae (Lohaus and Moellers 2000). The asparagine transamination pathway may also compensate for the absence of asparaginases (Zhang et al. 2012, Zhang and Marsolais 2014). Legumes predominantly express K+ -dependent asparaginase genes. Downregulation of K+ -dependent asparaginase in Lotus japonicus mutants has revealed that the gene is required for pod and seed development


and accumulation of storage proteins, likely by contributing to the assimilation of nitrogen transported as asparagine (Credali et al. 2013). The objective of this study was to evaluate whether asparaginase overexpression would lead to reduced steady-state levels of free asparagine in the developing embryo, and in turn whether seed protein concentration would be affected. To this end, a P. vulgaris cDNA coding for K+ -dependent asparaginase 2 (PvAspG2) was expressed in transgenic soybean under the control of the embryo-specific, seed storage protein promoter of the 𝛽-subunit of 𝛽-conglycinin, and several transgenic lines were characterized.

Materials and methods Generation of constructs and transgenic lines PvAspG2 (previously referred to as L-asparaginase 2) (Bruneau et al. 2006) was introduced under the control of the soybean embryo-specific 7S globulin promoter of the 𝛽-subunit of 𝛽-conglycinin (p𝛽C79) (Fujiwara et al. 1991) into the pCAM1302tCZ-PrxN-GUS vector backbone (Ivanov et al. 2012). A control construct with 𝛽-glucuronidase (GUS) (uidA) driven by the same promoter was also generated. For the experimental construct, the p𝛽C79 fragment was excised with HindIII and BamHI. The PvAspG2 cDNA was amplified by polymerase chain reaction (PCR) with AccuPrime Pfx DNA Polymerase (Life Technologies, Burlington, Canada) to generate BglII and BstEII sites at the 5′ and 3′ -end, respectively, using the following primers: PvASN-pBC79F, 5′ -AATTAGATCTATGGGGGGTTGGG CTAT-3′ , and PvASN-pBC79R1, 5′ -AATTGGTNACCTTA TTCCCAGATTCCAACC-3′ . The PCR product was cloned into the pCR BluntII TOPO vector (Life Technologies), verified by sequencing with a 3130XL Genetic Analyzer (Life Technologies), and digested with BglII and BstEII. The pCAM1302tCZ-PrxN-GUS vector was digested with HindIII and BstEII. The vector, promoter and cDNA fragments were ligated with T4 DNA ligase. For the control construct, the plasmid containing p𝛽C79 was digested with BamHI, ligated to a BamHI-NcoI linker-adapter (5′ -GATCCCATGG-3′ ) using T4 DNA ligase, and subsequently digested with HindIII and NcoI. The pCAM1302tCZ-PrxN-GUS vector was digested with the same restriction enzymes. The vector and promoter fragments were ligated with T4 DNA ligase. Transgenic soybean plants were generated following the protocols of Santarem and Finer (1999) (Fig. S1, Supporting Information). Somatic embryos were initiated on immature cotyledons of the Jack cultivar (Nickell et al. 1990), proliferated and subjected to microparticle 127

bombardment using a particle inflow gun constructed by Brown et al. (1994). Transgenic somatic embryos (T0) were selected in liquid Finer–Nagasawa Lite (FNL) media containing hygromycin (Samoylov et al. 1998). Genomic DNA was isolated from transformed embryos using Nucleon Phytopure Genomic DNA Extraction Kit (GE Healthcare Life Sciences, Baie d’Urfé, Canada) and the presence of the transgene was confirmed by PCR with primers for the selectable marker (hpt2), HYG1, 5′ -CATGTGTATCACTGGCAAAC-3′ , and HYG2, 5′ -CCAAGCTCTGATAGAGTTGG-3′ . Multiple somatic embryos were then generated for each separate transgenic event to provide tissue for assessment of transgene expression prior to plant regeneration. Transformed somatic embryos were screened for PvAspG2 transcript expression by reverse transcriptionpolymerase chain reaction (RT-PCR) using the gene-specific primers PvASNase2-F, 5′ -GTTGGATACG ACAGTTGCGGTGCGGG-3′ , and PvASNase2-R, 5′ -AG CCTCCTGCAGTTGCAGTCCCTTG-3′ . RNA was extracted using a modified lithium chloride method (Bruneau et al. 2006). The concentration and purity of the RNA samples was verified with a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE) and by agarose gel electrophoresis. The Superscript First-Strand Synthesis System for RT-PCR (Life Technologies) was used to make cDNA from 1 μg of total RNA. RT-PCR was carried out in a final volume of 25 μl, using 2 μl of cDNA template. Soybean isoflavone synthase 1 (IFS1) was used as control for RNA input with the following primers, GmIFS1-F, 5′ -CTCGGCGAGGCTG AGGAG-3′ , and GmIFS1-R, 5′ -ACTCTGTTGCCACCGCT GTG-3′ (Thibaud-Nissen et al. 2003, Dhaubhadel et al. 2007). Lines having high transcript levels of the transgene were selected for regeneration. Leaf samples from individual T0 plants were genotyped for the presence of the PvAspG2 or uidA transgene by PCR. Genomic DNA was extracted from leaf discs using a cetyl trimethylammonium bromide method (Richards et al. 2001). For PvAspG2, the PvASNase2-F and -R primers were used. For uidA, the following primers were used: GUS-F, 5′ -TAGAAACCCCAACCCGTGAAA-3′ , and GUS-R, 5′ -GAGTTTACGCGTTGCTTCCG-3′ . PvAspG2 transgene expression in developing T1 seeds of approximately 150 mg fresh weight was confirmed by RT-PCR. To screen for a control, transgenic lines expressing GUS, developing seeds undergoing desiccation or mature seeds (T1 generation) were collected from several different lines. Protein was extracted from a single seed and GUS activity was measured in extracts using a fluorometric assay (Inaba et al. 2007). Protein concentration in the extracts was measured using the Bio-Rad Protein Assay solution (Mississauga, Canada) and bovine serum 128

albumin as standard. GUS activity was determined using 1 mM 4-methyl-umbelliferyl-𝛽-D-glucuronide (MUG) as substrate. Production of 4-methylumbelliferone (MU) was monitored kinetically with a Hitachi F-2000 fluorescent spectrophotometer (Rexdale, Canada), using MU as standard (excitation wavelength 365 nm and emission wavelength 455 nm). Extracts from untransformed Jack seeds were used as controls. Plant growth and experimental design for transgenic experiments Plants were grown in growth cabinets (Environmental Growth Chambers, Chagrin Falls, OH) as previously described (Pandurangan et al. 2012). Plants were given 16 h light (300–400 μmol photons m−2 s−1 ) using 40 W A19 incandescent (OSRAM Sylvania, Mississauga, Canada) and ALTO F32T8/TL830 PLUS (Philips Lighting Canada, Markham, Canada) fluorescent lamps, and 8 h dark, with a temperature cycling between 18 and 24∘ C. At the beginning of the reproductive cycle, short day conditions were established by reducing the photoperiod to 12 h to boost flowering. To characterize transgenic lines, seeds (T1) from three independent lines having high transcript levels of the PvAspG2 transgene were sown alongside one control transgenic line having the highest GUS activity. Individual plants were genotyped and nontransgenic individuals discarded. Approximately six plants from each line were grown to maturity. Expression of the transgenes was confirmed by RT-PCR in small pools of developing seeds. The position of plants was randomized every 2 weeks. For each transgenic line, seeds randomly pooled from different individuals were used for characterization. For analysis at mid-maturation, replicate samples were composed of 15 developing seeds, while at maturity, a sample consisted of approximately 35 seeds. Developing seeds were harvested at approximately 9 h during the photoperiod. Amino acid analysis Extraction and quantification of free amino acids from seed tissues was performed as previously described, using HPLC after derivatization with phenylisothiocyanate (Hernández-Sebastià et al. 2005, Taylor et al. 2008). Generation of anti-asparaginase antibodies ARB-PVASPG1 rabbit polyclonal antibodies were raised against purified recombinant PvAspG2. Antibodies were raised and purified against the antigen by Biomatik (Cambridge, Canada). PvAspG2 cDNA was cloned into the PCR BluntII TOPO vector by RT-PCR


from RNA isolated from developing seeds of common bean cultivar AC Compass (Park and Rupert 2000), using the following primers: PvASNase1-NcoI-sense, 5′ -CTACCCATGGGAGGTTGGGCAATTG-3′ , and PvASNase1-XbaI-anti, 5′ -GTAGTCTAGATTAATCCCAAATTG CAACC-3′ . The insert was verified by sequencing. The insert was digested with NcoI and XbaI and subcloned into the same restriction sites of the pPROEX-HT vector (Life Technologies). For protein expression, the resulting construct was transformed into Escherichia coli OneShot BL-21(DE3) cells (Life Technologies). Protein was expressed and purified using an ÄKTAfplc system (GE Healthcare Life Sciences) as previously described (Gabriel et al. 2012). Affinity purified fractions containing PvASPG2 were pooled and precipitated by adding ammonium sulfate to 25% (w/v) saturation. The suspension was centrifuged at 20 000 g for 20 min at 4∘ C. The pellet was resuspended in 20 mM Tris–HCl, 10 mM KCl, pH 7.4 and desalted on a PD-10 column (GE Healthcare Life Sciences) equilibrated in the same buffer. Tobacco etch virus protease digestion was carried out in 1× AcTev buffer containing 1 mM dithiothreitol (DTT) and 5000 units of AcTev protease (Life Technologies) and 10 mg of recombinant protein. Samples were incubated at 16∘ C for 6 h. Reaction efficiency was determined to be 100% by running both uncleaved and cleaved samples on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. The protein was separated away from AcTev protease and the polyhistidine tag by chromatography on a HiLoad Superdex 200 16/60 prep grade column (GE Healthcare Life Sciences) in 20 mM Tris–HCl pH 7.5, 10 mM KCl. The eluted sample was concentrated with an Amicon Ultra-4 centrifugal filter unit (EMD Millipore, Billerica, MA). The protein was flash frozen in liquid nitrogen and stored at −80∘ C in the presence of 20% (v/v) glycerol. Extraction, assay and immunodetection of asparaginase Asparaginase was extracted and assayed from seed tissue as previously described (Pandurangan et al. 2012). The aspartate produced was measured by high pressure liquid chromatography (HPLC) after derivatization with o-phthalaldehyde and 3-mercaptopropionic acid, using a system consisting of a Waters 600E controller, 717 Plus Autosampler (Mississauga, Canada), Shimadzu RF-551 fluorescence detector (Columbia, MD), operated with EMPOWER 2 software. For immunodetection, protein in soluble extracts was separated by SDS-PAGE and transferred to a nitrocellulose membrane (9 × 6 cm) at 15 V for 20 min using a semi-dry transfer apparatus (Bio-Rad Laboratories). The membrane was blocked with


blocking buffer for fluorescent Western blotting (Rockland, Gilbertsville, PA) at room temperature for 1 h. The membrane was incubated with 1:5000 dilution of the primary antibody for 1 h, followed by a dilution of IRDye800® Conjugated Affinity Purified Anti-Rabbit IgG (goat) (Rockland) for 1 h. Immunodetection was achieved by scanning with an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE). Elemental analysis Samples were ground to a fine powder in a Cyclone Sample Mill (Udy Corporation, Fort Collins, CO) and freeze dried. Approximately 500 mg of ground tissue was submitted to elemental analysis which was performed by dry combustion with a LECO CNS-2000 Elemental Analyzer (Mississauga, Canada) as described by Taylor et al. (2008). Oil quantification Ground tissue (2 g) was placed in an Erlenmeyer flask; 20 ml of hexane was added and the flask was agitated on an orbital shaker at 70 r.p.m for 1 h. The hexane was decanted and discarded. In total, this extraction was repeated three times. The defatted meal was air-dried overnight, and the weight of the flask recorded. Globulin and albumin extraction and quantification Albumin and globulin fractions of extractible proteins from mature seed were determined as described by Rolletschek et al. (2005). Protein was extracted from 100 mg of defatted meal using 1 ml of extraction buffer. Band intensities in globulin extracts were measured with the QUANTITY ONE software (Bio-Rad Laboratories). Apparent molecular mass was measured with the same software. Statistical analysis ANOVA was performed using the SUPERANOVA statistical program (Abacus Concepts, Berkeley, CA) and SAS version 9.2 (Toronto, Canada). Homogeneity of the variances was evaluated by Bartlett’s test. Means were compared using Tukey’s range test at P ≤ 0.05 and P ≤ 0.01.

Accession numbers Accession number of PvAspG1 and PvAspG2 in the early release genome of the common bean (http://www. phytozome.org) is as follows: Phvul.001G02500.1 and Phvul.009G067800.1, respectively (Schmutz et al. 2014). 129

Results Production of transgenic soybean plants Transgenic plants were generated by microparticle bombardment of somatic embryos and by plant regeneration (Fig. S1). Embryos were transformed with a fusion between the embryo-specific promoter of the 𝛽-subunit of 𝛽-conglycinin, p𝛽C79 (Fujiwara et al. 1991) and the PvAspG2 cDNA in the pCAM1302tCZ-PrxN-GUS vector backbone (Ivanov et al. 2012). This vector has been designed to prevent interference between a constitutive promoter driving expression of a selectable marker and a tissue-specific promoter (Yoo et al. 2005, Zheng et al. 2007, Gudynaite-Savitch et al. 2009). Heterologous expression of the P. vulgaris PvAspG2 cDNA was selected in order to avoid co-suppression. The fact that the gene encoding 𝛽-subunit of 𝛽-conglycinin is actively transcribed in somatic embryos (Thibaud-Nissen et al. 2003) enabled screening of liquid cultured transformed embryos for high expression of the PvAspG2 transgene by RT-PCR. Clones with high level expression were selected for regeneration. Transgene expression was confirmed by RT-PCR in the T1 seed progeny of the regenerated plants at 150 mg seed weight. This stage was selected because it is close to the one at which free amino acids were analyzed in earlier studies (100 mg seed weight) (Hernández-Sebastià et al. 2005, Pandurangan et al. 2012) and coincides with the onset of the

accumulation of the 𝛽-subunit of 𝛽-conglycinin protein (Gayler and Sykes 1981), while the corresponding transcript is readily detectable (Meinke et al. 1981, Naito et al. 1988). Three independent lines expressing high levels of the PvAspG2 transgene were selected and successfully regenerated, and designated B, E and I. A control line was used expressing high levels of a neutral protein, the reporter enzyme GUS, encoded by uidA (Jefferson et al. 1987). A large number of independent transgenic lines were screened for GUS activity in extracts from seed at late maturation or maturity. The line having the highest activity, designated P150-34, was selected as control. GUS activity in this line was equal to 2.65 pkatal mg−1 protein (average of duplicate measurements).

Characterization of asparaginase expression in transgenic lines T2 seeds were used for characterization. Transgene expression in developing seeds of 150 mg was confirmed at this generation. Fig. 1A shows RT-PCR results indicating that transcript expression of the PvAspG2 transgene was specific to the B, E and I lines, whereas the uidA transcript was detected exclusively in the control line. Expression of the PvAspG2 protein was evaluated by immunoblotting. Asparaginase in soluble extracts was immunodetected with polyclonal antibodies raised against a purified recombinant PvAspG2 expressed in

Fig. 1. Characterization of transgene expression in soybean lines. (A) RT-PCR analysis of transcript expression in transgenic lines expressing PvASPGB1, B, E and I and in the control line expressing uidA, P150-34, under the control of promoter of the 𝛽-subunit of 𝛽-conglycinin. Binary vector plasmids were used as positive controls for PCR amplification. The untransformed Jack cultivar was used as negative control. IFS1 was used as positive control for RT-PCR amplification. NTC: no template control. (B) Immunoblot of soluble protein extracts containing asparaginase probed with polyclonal antibodies raised against PvASPG2. Size of markers is indicated on the left. Position of 𝛼- and 𝛽-subunits is indicated on the right by arrows.

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Table 1. Asparaginase activity in cotyledon extracts from seeds of approximately 150 mg from transgenic control and experimental lines expressing PvASPGB1. Activity is expressed in pkatal per mg protein. n = 3; CV: coefficient of variance; values with a common letter are not significantly different according to Tukey’s range test at P ≤ 0.01; **** denotes ANOVA P value ≤0.0001. Line P150-34 B E I CV (%) F-value

Asparaginase activity 0.21c 5.03b 8.63a 4.37b 10.1 168****

E. coli. A band was detected at approximately 23 kDa, corresponding to the 𝛼-subunit, having a predicted molecular mass of 21 kDa (Fig. 1B). The signal from this band was the most intense in line E, intermediate in lines B and I, and much lower in the control line, where it likely represents the detection of endogenous GmASPGB1 and GmASPGB2 isoforms, whose transcripts are expressed in the developing embryo (Pandurangan et al. 2012). A lower band at approximately 14 kDa was detected, corresponding to the 𝛽-subunit, having a predicted molecular mass of 13.5 kDa. The signal for this band was also of the highest intensity in line E and was lower in lines B and I. Asparaginase activity was quantified in soluble protein extracts from developing cotyledons and expressed in pkatal per mg protein. Activity was 20- to 40-fold higher in the transgenic lines expressing PvAspG2 than in the control line (Table 1). Line E had the highest activity, approximately twofold higher than line B or I, consistent with the results of immunoblotting. Effects on free amino acids in developing seed To evaluate whether PvAspG2 expression has an effect on the levels of free asparagine and other free amino acids in developing seeds, cotyledons from seeds of approximately 150 mg were extracted and free amino acids quantified by HPLC. The concentration of total free amino acids was not uniform between lines. Line B had approximately one third less of total free amino acids as compared with the three other lines (Table S1). Asparagine levels were significantly decreased in the three lines expressing PvAspG2 as compared with the control line, expressed as a percentage of total free amino acids (Fig. 2 and Table S2). Free asparagine concentration was equal to 48% of total free amino acids in the control line as compared with 40–43% in the transgenic lines expressing PvAspG2. Conversely, the levels of free aspartic acid, the product of asparaginase, were raised


from 3.0% of total free amino acids in the control line to 3.8–4.9% in the transgenic lines expressing PvAspG2. The proportion of nitrogen-rich amino acids, arginine and lysine, and of the major intermediates in nitrogen flux in amino acid metabolism, serine and alanine was elevated in all of the lines expressing PvAspG2 as compared with the control. The only other free amino acid beside asparagine with reduced levels in all of these lines as a percentage of total free amino acids was the arginine precursor citrulline. The proportion of some free amino acids was raised significantly in lines E and I, but not in line B as compared with the control, including the hydrophobic amino acids leucine, valine, isoleucine and phenylalanine, and methionine and glutamine, the proportion of the latter being raised by 11–30%. A higher percentage of glutamic acid, raised by 42%, clearly differentiated line B from the control and the two other lines expressing PvAspG2. Effects on the composition of mature seed Mature seeds were analyzed for total carbon, nitrogen and sulfur concentration by elemental analysis to evaluate potential effects on seed protein concentration. Two of three lines expressing PvAspG2, E and I, had reduced nitrogen concentration as compared with the control line, by 9–13%, whereas the carbon and sulfur concentration remained unchanged (Table 2). The reduction in nitrogen concentration was paralleled by an increase in oil concentration, by 5–8%. The reduction in nitrogen concentration and increase in oil concentration were most pronounced in line E, which has higher asparaginase activity than line I. These changes in seed composition were not related to a difference in seed size, as individual seed weight was similar between lines, expressed in mg (average ± standard deviation): for P150-34, 153 ± 10; B, 147 ± 14; E, 158 ± 3; I, 146 ± 15 (ANOVA P value not significant; n = 8 plants). Albumins and globulins were extracted from mature seed and their concentration compared between lines. The levels of albumins were similar, but lines E and I had a lower concentration of globulins, by 17–28% as compared with the control (Table 3). As with nitrogen concentration, the change in globulin concentration was more pronounced in line E than line I. The major protein bands in globulin extracts have a known mobility in SDS-PAGE (Thanh and Shibasaki 1977, Staswick et al. 1981) which has been confirmed by proteomic information (Mooney and Thelen 2004). Protein profiles were examined by subjecting to SDS-PAGE a volume of sample equivalent to a similar weight of mature seed tissue extracted (Fig. 3), and globulin bands were quantified by image analysis. In line E, all major bands appeared to be reduced (Table 4). In line I, 131

Fig. 2. Free amino acid concentration as a percentage of total free amino acids in developing cotyledons at approximately 150 mg seed weight from transgenic control and experimental lines expressing PvASPGB1; error bars represent standard deviation; n = 4;ANBA: amino-N-butyric acid. Table 2. C, N, S and oil concentration in mature seed of transgenic control and experimental lines expressing PvASPGB1, expressed as percent of seed weight. n = 4; each n replicate is the average of technical duplicates; CV: coefficient of variance; n.s.: not significant; values with a common letter are not significantly different according to Tukey’s range test at P ≤ 0.01 for N and P < 0.05 for oil; **** ANOVA P ≤ 0.0001. Line P150-34 B E I CV (%) F-value

C 53.3 53.6 53.6 53.5 0.55 0.66n.s.

N

5.54a 4.79b 5.05b 2.36 36.0****

the levels of the 𝛼 ′ - and 𝛼-subunits of 𝛽-conglycinin and glycinin A subunits were reduced, whereas those of the 𝛽-subunit of 𝛽-conglycinin and glycinin B subunit were similar to the control. As expected, levels were similar between line B and the control.

Discussion A correlation has been previously observed between the levels of free asparagine in the developing embryo and protein concentration in mature soybean seed (Hernández-Sebastià et al. 2005, Pandurangan et al. 2012). In this study, asparaginase was overexpressed in the developing embryo to test whether this would alter the steady-state concentration of free asparagine, and seed protein concentration. Expression of PvAspG2 under the control of the promoter of the 𝛽-subunit of 𝛽-conglycinin effectively raised the levels of asparaginase activity in the developing embryo (Table 1). However, in the control line, the levels of GUS activity in seed were comparable to those achieved with the tobacco tissue specific ASA2 promoter, but approximately one-thousandth of that with the CaMV35S promoter (Inaba et al. 2007). The p𝛽C79 fragment used in this study constitutes a 1.1 kb proximal promoter of the 𝛽-subunit of 𝛽-conglycinin (Fujiwara et al. 1991). Although it drove GUS reporter expression at levels comparable to those of the CaMV35S promoter in 132

S

5.54a

0.255 0.258 0.258 0.265 4.72 0.45n.s.

Oil 18.32c 18.62c 19.89a 19.28b 1.30 31.7****

protoplasts isolated from soybean cultured cells, the present results suggest that it lacks the regulatory elements required for full strength expression in developing seeds. Overexpression of asparaginase effectively lowered the steady-state levels of free asparagine, while it raised those of free aspartic acid in developing cotyledons at mid-maturation in three independent transgenic lines, expressed as a percentage of total free amino acids (Fig. 2 and Table S2). The reduced concentration of nitrogen in mature seed in two of these lines suggests that increasing asparaginase expression can lower seed protein concentration (Table 2). The reduced nitrogen concentration was associated with a specific decrease of the extractible globulin protein fraction (Tables 3 and 4, Fig. 3). These changes were paralleled by an increase in total oil concentration indicating a modified partitioning of carbon between storage products (Table 2). Effects on mature seed composition were correlated with the strength of asparaginase expression in lines E and I (Table 1, Fig. 1). Line B, which did not exhibit changes in mature seed composition was characterized by a large increase in the relative concentration of free glutamic acid at mid-maturation, as compared with the other lines (Fig. 2). These results suggest that the relative increase in free glutamic acid concentration may compensate for the decreased proportion of free asparagine. Glutamic acid or a glutamic acid-derived metabolite has been


Table 3. Concentration of extractible albumins and globulins in mature seeds of transgenic control and experimental lines expressing PvASPGB1 expressed in percent of seed weight. n = 4; each n replicate is the average of technical duplicates; CV: coefficient of variance; n.s.: not significant; values with a common letter are not significantly different according to Tukey’s range test at P ≤ 0.05; **** ANOVA P ≤ 0.0001. Line P150-34 B E I CV (%) F-value

Albumins 1.78 1.97 1.72 1.88 7.54 2.66n.s.

Globulins 11.1a 11.0a 7.76c 9.88b 4.31 51.6****

Fig. 3. SDS-PAGE of globulin extracts from mature seeds of control line expressing uidA, P150-34, and transgenic lines expressing PvASPGB1, I (A) and B and E (B) and under the control of promoter of the 𝛽-subunit of 𝛽-conglycinin. A volume of sample equivalent to the same tissue weight extracted was subjected to SDS-PAGE. Size of markers is indicated on the left. Position of the 𝛼 ′ -, 𝛼- and 𝛽-subunit of 𝛽-conglycinin and acidic (A) and basic (B) subunit of glycinin is indicated on the right.

hypothesized as a putative organic nitrogen signal in Arabidopsis seedlings (Gutiérrez et al. 2008). Alternatively, the possibility that the transgene disrupts some endogenous gene in line B, resulting in a different phenotype cannot be excluded. The decrease in nitrogen concentration in lines E and I is unlikely to result from competition between the promoters of the transgene and of the endogenous 𝛽-subunit gene, given the relatively low GUS expression Physiol. Plant. 155, 2015

driven by the p𝛽C79 promoter, as discussed above. Increased expression of asparaginase may alter the flux of asparagine into protein or the flux of nitrogen from asparagine into other amino acids. While free asparagine accounts for close to 50% of total free amino acids in developing cotyledons at mid-maturation, the combined concentration of total asparagine and aspartic acid measured after acid hydrolysis in mature seed of Jack accounts for 12% of total amino acids in mg per g dry weight, as compared with 18–19% for glutamine and glutamic acid (Dinkins et al. 2001, Kita et al. 2009, Ishimoto et al. 2010). In comparison, glycinin and 𝛽-conglycinin subunits contain 6–10% of their amino acid residues as asparagine, and 3–5% as aspartic acid, 5–14% as glutamic acid and 7–12% as glutamine (Doyle et al. 1986, Harada et al. 1989, Nielsen et al. 1989, Sebastiani et al. 1990). These data indicate that the reduction in globulin concentration in lines E and I is probably not related to the concentration of asparagine in globulins. The reduction in globulins might happen as a response to changes in the levels or flux of free amino acids in the transgenic lines. Transcript levels of the 𝛽-subunit of 𝛽-conglycinin have been shown to be regulated by exogenous glutamine in developing seeds grown in vitro (Ohtake et al. 2002, Hernández-Sebastià et al. 2005). Ohtake et al. (2002), using treatments with the amides glutamine or asparagine in combination with inhibitors of aminotransferases or glutamate synthase, concluded that the transcript of the 𝛽-subunit is highly sensitive to nitrogen status, is not regulated by methionine, glutamine or asparagine in isolation, and that it may respond to a glutamine metabolite. Changes in the relative levels of free amino acids in developing seeds can be interpreted as a metabolic adaptation to the effects of transgene expression. Some changes were observed in all three lines expressing asparaginase (Fig. 2). The increase in the proportion of the nitrogen-rich amino acids, arginine and lysine, might be a compensatory mechanism for the decrease in asparagine. Serine and alanine are central intermediates in amino acid metabolism and shared substrates of asparagine transaminase in Arabidopsis (Zhang et al. 2012). If catabolism of asparagine by transamination was lowered in response to asparaginase expression in the transgenic lines, one would predict that the relative levels of serine and alanine would be elevated. The increased proportion of branched chain amino acids, of the aromatic amino acid phenylalanine, of methionine and of glutamine in lines E and I likely reflects an adaptation to the decrease in the relative concentration of asparagine, in the absence of a change in glutamic acid as in line B. These amino acids are linked metabolically. Phenylalanine is derived from phosphoenolpyruvate and branched 133

Table 4. Relative intensity of protein bands in globulin extracts from control and experimental lines expressing PvAspG2 in Fig. 3. n = 4; molecular mass in kDa (average ± standard deviation): 𝛼 ′ -subunit of 𝛽-conglycinin, 89.6 ± 0.6; 𝛼-subunit of 𝛽-conglycinin, 79.4 ± 0.7; 𝛽-subunit of 𝛽-conglycinin, 51.0 ± 0.3; acidic (A) subunit of glycinin, 35.5 ± 0.2; basic (B) subunit of glycinin, 16.4 ± 0.1; CV: coefficient of variance; values with a common letter in each column are not significantly different according to Tukey’s range test at P ≤ 0.05; ANOVA P **** ≤ 0.0001; *** ≤ 0.001; ** ≤ 0.01. Line P150-34 B E I CV (%) F-value

𝛼-subunit of 𝛽-conglycinin a

1.00 1.03a 0.71b 0.68b 12.56 11.79****

𝛼 ′ -subunit of 𝛽-conglycinin a

1.00 0.94a 0.67b 0.70b 12.11 10.78***

chain amino acids from pyruvate. Both isoleucine and methionine are part of the aspartic acid family of amino acids, and the branched chain amino acids and methionine are related in their metabolism and regulation through methionine 𝛾-lyase (Joshi et al. 2010). It was recently shown that Arabidopsis transgenics expressing feedback insensitive cystathionine 𝛾-synthase in an embryo-specific manner have elevated levels of free amino acids in mature seed (Cohen et al. 2014). These changes were associated with an increased concentration of total amino acids and nitrogen in mature seed, and enhanced ethylene and abscisic acid signaling during seed development. Expression of the same transgene in soybean seed led to increased soluble methionine concentration in three independent transgenic lines (Song et al. 2013). Two of these lines had an increased accumulation of total methionine and total amino acids, resulting in increased protein concentration. It was also recently demonstrated that enhanced production of 𝛾-aminobutyric acid (GABA) during seed desiccation in transgenic Arabidopsis changed the balance of storage products, resulting in increased protein and decreased oil concentration (Fait et al. 2011). These changes were achieved by embryo-specific expression of a glutamate decarboxylase lacking its regulatory Ca2+ -calmodulin-binding domain under the control of the 7S globulin phaseolin promoter. The elevated protein concentration in the transgenic Arabidopsis lines was attributed to increased availability of free amino acids, including aspartate-derived amino acids, serine, cysteine, methionine and tryptophan, associated with enhanced GABA production. This study offers another example where an alteration of amino acid metabolism in the developing embryo can result in changes in seed protein concentration. The findings presented here advance our knowledge and understanding of the control of protein reserve accumulation by showing that overexpression of asparaginase in the developing embryo results in a decrease in the relative concentration of free asparagine, which can be associated with a reduction in 134

𝛽-subunit of 𝛽-conglycinin

Glycinin A subunit

a

1.00 1.08a 0.73b 0.92a 8.94 13.16****

a

1.00 1.07a 0.74b 0.80a 9.12 13.64****

Glycinin B subunit 1.00a 0.97a 0.81b 0.97a 7.21 5.87**

seed nitrogen concentration, rather than increasing the flux of nitrogen into storage proteins.

Author contributions S. P., A. P., D. C. W. B. and F. M. designed the research; S. P., A. P. and T. R. performed experiments; R. B. provided access to and supervised the use of an elemental analyzer; S. P., A. P., T. R., R. B., C. H. S., D. C. W. B. and F. M. analyzed data; S. P., A. P., T. R., C. H. S., D. C. W. B. and F. M. wrote the manuscript. Acknowledgements – We thank Luanne Bruneau for assistance with promoter cloning, Tim McDowell for elemental analysis, Alex Molnar for preparation of figures, Yuhai Cui for providing the p𝛽C79 plasmid, Marwan Diapari for support with statistics, and Rey Interior, at the SPARC BioCentre, Hospital for Sick Children, Toronto, Ontario, for help with amino acid analyses. We are grateful to Denis Maxwell, from the University of Western Ontario, for serving as SP’s co-supervisor during his MSc program.

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Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. Flowchart explaining the generation and selection of soybean transgenic lines. Table S1. Concentration of free amino acids in developing cotyledons expressed in nmol mg−1 . Table S2. Concentration of free amino acids in developing cotyledons expressed in percentage of total free amino acids.

Edited by A. Marion-Poll


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