Chemical characterization of autoclaved products
The yield of GlA20 and GlA40 was found in the range of 92-95%. While GlA20 was observed in white crystalline powder, GlA40 was a creamy white powder. The latter was highly hygroscopic and turned into a pale yellow sticky mass within an hour under ambient conditions.
Thin Layer Chromatography
TLC analysis of GlA20, GlA40 and GFs showed distinct differences in their Rf values. GlA20 showed only one spot at Rf 0.73, hence, confirmed the formation of only one product (II). However, in case of GlA40, one major spot was observed at Rf 0.70 together with a minor spot at Rf 0.73. The observation suggested the presence of one major compound (III) along with a minor compound (II) in GlA40. The difference in the Rf values of both products from GFs (Rf 0.77) indicated the transformation of L-glutamine upon autoclaving.
NMR and mass spectroscopy
The NMR spectral data (1H, 13C and 2D experiments) provided evidence for GlA20 and GlA40 being two structurally different compounds (Figure ).
NMR spectral data (1H, 13C and 2D experiments) of L-glutamine and its autoclaved products (GlA20 and GlA40).
GlA20 was identified as 5-oxo proline (pyroglutamic acid, II) on the basis of comparison of NMR and mass spectral data with reported values. Electrospray ionization mass spectrometry (ESI-MS) of GlA20 showed protonated molecular ion at m/z 130 [M+H]+ which corresponded to the molecular formula C5H7NO3.
13C NMR spectrum of GlA40 was dominant with the signals of major product (III) that showed 5 carbon resonances, of which two are observed at δ 182.1 and 180.0 for carbonyls. A downfield shift of these resonances when compared with parent compound (control) indicated presence of a cyclic ring. The other 3 carbons resonated at δ 58.2, 29.9 and 25.5. Considerable shifts in these resonances in comparison to the respective resonances of control (L-glutamine) provided further support for the cyclic structure for product III. DEPT 135 experiment revealed the nature of these three resonances as two CH2 at δ 29.9 and 25.5 and one as CH at δ 58.2. In 1H NMR spectrum, three multiplets were observed at δ 1.93, 2.38 and 4.08, each integrating for two, two and one proton, respectively. The multiplet patterns of these three proton resonances due to mutual scalar couplings indicate that they are connected in series. The coupling pattern was further confirmed by COSY. Electrospray ionization mass spectrometry (ESI-MS) of GlA40 showed protonated molecular ion at m/z 129 [M+H]+ for major product (III) which corresponded to the molecular formula C5H8N2O2. The structural elucidation of compound III finally revealed complete cyclization of glutamine during autoclaving. HMQC and HMBC study further confirmed the loss of a molecule of water to form compound III i.e. α-amino glutarimide or 3-amino-2,6-piperidinedione (Figure and ).
(a) Chemical transformation of L-glutamine into pyroglutamic acid and 3- or α-amino glutarimide upon autoclaving at 121°C and 15 psi (b) Key HMBC of α-amino glutarimide.
The results clearly suggested that the formation of α-amino glutarimide (III) was preferred over pyroglutamic acid (II) when L-glutamine was over autoclaved.
α-amino glutarimide: 1H NMR δ: 1.93 (m, 2H), 2.38 (m, 2H), 4.08 (q, 1H); 13C NMR δ: 182.1, 180.0, 58.2, 29.9, 25.5; ESI-MS m/z: 129 [M+H]+.
Agrobacterium growth in response to autoclaved products of L-glutamine
A. tumefaciens growth was more pronounced at pH 7.0 as compared to acidic pH (5.2 to 5.9), irrespective of supplements (GFs, GlA20, 5-oxo proline or GlA40,), and a similar trend was recorded in both YMB and MS (Figure ). However, growth was remarkably higher in YMB as compared to MS. As compared to control, maximum growth was recorded at pH 7.0 in the presence of GlA40 (i.e, 3.0 and 2.68 fold in YMB and MS, respectively). While growth in the presence of GlA20 was 1.2 and 0.8 fold, that in the presence of 5-oxo proline was about 1.0 and 0.6 fold in YMB and MS, respectively. Growth in the presence of GFs was always lower than control, irrespective of culture media. At acidic pH of YMB ranging from 5.2 to 5.6, growth was lower than that in the presence of GFs (0.5 to 0.54 fold), GlA20 (0.81 to 0.89 fold) and 5-oxo proline (0.88 fold). However, about 1.4 fold growth was recorded in the presence of GlA40 (Figures and ). In case of MS, a trend similar to that in YMB was observed. While growth was lower by 0.5 fold in the presence of GFs, it ranged between 0.75 to 0.88 fold in the presence of GlA20 and 5-oxo proline. However, growth in presence of GlA40 was 1.3 to 1.4 fold.
A. tumefaciens growth in (a) YMB and (b) MS as represented by OD × 1 × 109 cfu ml-1 at A600 nm in response to 2.0 g l-1 GFs, GlA20, GlA40 and control.
Effect of pH (5.2-5.9 and 7.0) on A. tumefaciens growth and GUS expression of explants transformed in presence of GFs, GlA20 and GlA40.
β-galactosidase activity as a measure of Agrobacterium virulence (vir) gene induction
Considerably high β-galactosidase activity was recorded in the presence of AS, a known vir gene inducer. However, the activity of control (no additions) was lower than that in the presence of AS (Table ). While the activities were 132.7 and 132.0 Miller units in the presence of GFs and GlA20, respectively, suppression (130.9 Miller units) was observed in the presence of GlA40.
vir gene induction by L-glutamine (GFs) and its autoclaved products (GlA20 and GlA40)
Agrobacterium infection of resistant host plants by autoclaved products of L-glutamine
A. tumefaciens growth on leaf explants varied with plant species when transformed with different cell densities and co-cultivated on MS supplemented with GlA40. No growth was observed on any of the explants when transformed with 1 × 107 cfu ml-1 of A. tumefaciens followed by co-cultivation on MS containing either of GFs, GlA20 or 5-oxo proline. Slight A. tumefaciens growth was however, recorded on explants of bamboo, maize and apple rootstock transformed using a cell density of 1 × 107 cfu ml-1 followed by co-cultivation in the presence of GlA40. No explant, except tobacco showed A. tumefaciens growth when co-cultivated on control medium after transformation using cell densities up to 1 × 108 cfu ml-1. However, explants of apple rootstock, bamboo, maize, and rice showed slight growth on control medium at all tested cell densities beyond 1 × 108 cfu ml-1.
Growth increased with further increase in cell density i.e., lowest at 1 × 108 cfu ml-1 and highest at 1 × 1010 cfu ml-1 in case of Indian may apple, aloe, lavender, wild rose, apple rootstock, bamboo, grass, maize and rice; and also in the leaf explants of fern and Aurocaria when co-cultivated on MS containing GlA40 (Figure ). Even in case of control i.e., in the absence of supplements, A. tumefaciens growth was observed on apple rootstock and rice when they were transformed using either of 1 × 109 or 1 × 1010 cfu ml-1. However, A. tumefaciens failed to grow on any of the explants when either GlA20 or 5-oxo proline was present in the co-cultivation medium, irrespective of cell densities, (Sigma, USA).
Figure 5 Agrobacterium growth on leaf explantsof; (a) tobacco, (b) grass, (c) Indian may apple, (d) rose, (e) bamboo and (f) fern (transformed using 1 × 109 cfu ml-1 followed by co-cultivation on MS containing GlA40 at pH 5.6); (g-h) growth in response (more ...)
When pH of the co-cultivation medium was considered, depending upon the plant species, slight to profuse growth was recorded on or around the explants in the acidic pH range only. In contrast, no A. tumefaciens growth was recorded on or around the explant surface at pH 7.0, irrespective of supplements.
GUS expression varied with A. tumefaciens density, plant species or presence or absence of GlA40 (Table ). Irrespective of the presence or absence of GlA40 in the co-cultivation medium, tobacco showed GUS expression at all cell densities ranging from 1 ×107 and 1 × 109 cfu ml-1. On the other hand, the presence of GlA20 or 5-oxo proline failed to induce GUS expression in all the studied plant species including tobacco.
GUS expression after 2 days of co-cultivation in leaf explants transformed using different A. tumefaciens population densities
GUS expression improved with increase in A. tumefaciens cell densities from 1 × 107 to 1 × 109 cfu ml-1 in the presence of GlA40 in case of apple rootstock, bamboo and maize; and from 1 × 108 to 1 × 109 cfu ml-1 in Indian may apple, aloe, lavender, grass and rice. However, the best response in terms of strong GUS expression spread over a larger area was recorded, only when 1 × 109 cfu ml-1 of A. tumefaciens was used, irrespective of plant species (Figure ). Increase in cell density beyond 1 × 109 cfu ml-1 had no effect on GUS expression (or in other words transformation) in case of aloe, grass, maize, rice, aurocaria and fern. On the other hand, with time, the explants turned necrotic due to A. tumefaciens overgrowth at 1 × 1010 cfu ml-1 in case of tobacco, Indian may apple, lavender, wild rose and apple rootstock.
Figure 6 GUS expression of leaf explants transformed using 1 × 109 cfu ml-1 followed by co-cultivation at pH 5.6. (a) tobacco, (b) grass (c) Indian may apple, (d) apple rootstock, (e) rose, (f) bamboo, (g) aurocaria (h) fern, (i) aloe, (j) lavender (k) (more ...)
The leaf explants of all the studied plant species showed GUS expression only in the acidic pH range when co-cultivated on MSC containing GlA40. However, the pH optima and the intensity of expression varied with the plant species tested (Table ). While the leaf explants of tobacco, lavender, apple rootstock, bamboo, maize and rice showed GUS expression at all pH in the acidic range (i.e., 5.2, 5.6 and 5.9), those of Indian may apple, wild rose and grass showed GUS expression at pH 5.6 and 5.9 only. On the other hand, the explants of aloe and fern tested positive when co-cultivated at pH 5.2 and 5.6, whereas, that in aurocaria was best observed at pH 5.6 only.
GUS expression in response to different pH during co-cultivation on MSC containing GlA40 after transformation of leaf explants of different plant species using 1 ×109 cfu ml-1 A. tumefaciens
In case of media containing 100 μM AS, but free of all glutamine supplements, longer co-cultivation time was required for the explants of wild rose, apple rootstock, bamboo, grass, maize, rice and fern (Table ). The only exception was tobacco where < 1 day of co-cultivation was sufficient for strong GUS expression. On the other hand, no GUS expression was observed in explants of any of the plant species co-cultivated at pH 7.0 (Table ; Figure ).
PCR confirmation of genetic transformation
PCR amplification products of about 490 bp corresponding to gus gene were observed in the leaf calli of aloe, lavender, tobacco, rose, grass, fern and apple rootstock and maize provided GlA40 was present in the co-cultivation medium (Figure and ). However, amplification was not detected in the calli derived from explants co-cultivated on either control or on media containing GFs, GlA20 or AS.
Figure 7 PCR of genomic DNA from leaf callus showing amplification of 490 bp gus gene. (a) Lane P: plasmid, Lane M: 0'GeneRuler™ 100 bp plus DNA ladder (Fermentas, Life Sciences), Lanes CA-AV: transformed aloe (CA: in absence of GlA40 and AV: in presence (more ...)
Genomic DNA of calli derived from aloe, wild rose, maize, fern, lavender and grass leaves transformed using A. tumefaciens density of 1 × 109 cfu ml-1 and co-cultivated in the presence of GlA40 tested positive in Southern hybridization and distinct purple-blue signals were detected (Figure ). However, no hybridization signals were observed in case of leaf calli obtained from explants co-cultivated in the presence of AS but in absence of GlA40 (Figure ). Hybridization signals were also not detected in the untransformed leaf calli of the studied plant species (not shown). Only tobacco and apple rootstock MM106 showed the hybridization signals both in the presence or absence of GlA40 in the co-cultivation medium (not shown). A distinct single band (> 3 kb) was observed in case of aloe (Figure , lanes 1 and 2). While a distinctly sharp band of > 3 kb was observed along with > 1.5 kb and 700 bp bands in wild rose (Figure , lane 3), four bands above 3 kb and two bands of about 1.8 kb and 700 bp were observed in maize (Figure , lane 4). No signal was observed in case of fern (Figure , lane 5) whereas, two sharp bands of about 1.8 kb and 700 bp were observed in case of lavender (Figure , lanes 6 and 7). Three distinct bands (400 bp, 900 bp and 2.8 kb) were observed in case of grass (Figure , lane 8).
Figure 8 Southern blot of genomic DNA from leaf callus hybridized with Biotin labeled gus gene probe. (a) Control Lanes 1-5: transformants in absence of GlA40 but presence of AS (1 and 2: aloe, 3: wild rose, 4: maize, 5: fern, 6: lavender, 7: grass (b) transformants (more ...)