The effect of bacterial isochorismate synthase on the Brassica rapa metabolome
Simoh, S.
Citation
Simoh, S. (2008, June 11). The effect of bacterial isochorismate synthase on the Brassica rapa metabolome. Retrieved from https://hdl.handle.net/1887/12944
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Chapter 7
1
H-NMR analysis of metabolomic changes in Brassica rapa upon infection with
Agrobacterium tumefaciens
Sanimah Simoh1,2, Hye Kyong Kim1, Young Hae Choi1, Robert Verpoorte1
1 Section Metabolomics, Institute of Biology, Leiden University, Leiden, The Netherlands
2 Biotechnology Research Centre, Malaysian Agricultural Research & Development Institute (MARDI), Kuala Lumpur, Malaysia
Abstract
The metabolic response of Brassica rapa to infection with disarmed and tumor-inducing strains of Agrobacterium tumefaciens was investigated using high resolution 1H-NMR spectroscopy combined with multivariate data analysis. Investigation of the score and loading plots of partial least square-discriminant analysis (PLS-DA) of the leaves infected with a disarmed strain (LBA4404) of A. tumefaciens suggested that there was a suppression of flavonoids (quercetin, kaempferol) and phenylpropanoids (trans- and cis- sinapoyl malate and coumaroyl malate) accumulation in comparison to the controls. A similar phenomenon was also observed for the leaves and stems taken from tumor- bearing plants infected with octopine and nopaline strains of A. tumefaciens. The present study also shows that octopine tumors induced by octopine strains have higher concentrations of the amino acids threonine, valine, leucine/isoleucine, the organic acids succinic acid, malic acid, formic acid, fumaric acid, and adenine and the flavonoid quercetin, whereas the tumors induced by nopaline strain have higher concentrations of the amino acids alanine, glutamic acid, and glucose, sucrose and phenylpropanoid derivatives.
Keywords: octopine, nopaline, Agrobacterium tumefaciens, NMR spectroscopy, Brassica rapa
7. 1 Introduction
Agrobacterium tumefaciens is a well known plant pathogenic bacterium that has the ability to deliver part of its genetic material, the T-DNA from its tumor-inducing (Ti) plasmid into the genome of the host plant, most of which are dicotyledonous. Plant tissue that has been infected with Agrobacterium possessing T-DNA synthesizes a crown gall tumor. Tumor–specific compounds called opines are formed as a result of the expression of the T-DNA gene in the tumor cell. Opines serve as a major carbon/nitrogen source for Agrobacterium (Bevan et al., 1982; Nester et al., 1984). They are reductive conjugates of L-amino acids and organic acids of primary metabolites e.g. pyruvate, α-ketoglutarate or glucose (Chilton et al., 2001). The type of opines produced is determined by the bacterial strains that incite the tumor. Octopine-, nopaline-, succinamopine- or leucinopine-specific strains exist (Hooykaas and Beijersbergen, 1994). The most common are octopine and nopaline strains which produce octopine (conjugate of arginine and pyruvate) and nopaline (conjugate of arginine and α-ketoglutarate) respectively.
The unique characteristic of Agrobacterium to transfer the T-DNA into a host plant is driven by the virulence genes which are located outside of the T-DNA region. The infection is regulated by a two component signal transduction system consisting of virulence proteins encoded by virA and virG genes. These proteins sense the chemical signals (monosaccharides and phenolic compounds such as acetosyringone) released by the wounded plant cell leading to the activation of other virulence genes. The expression of all these genes initiates the T-DNA transfer from the Agrobacterium to the host genome. Another component, a set of virulence (chv) genes located in the bacterial chromosome is involved in bacterial chemotaxis and attachment on the wounded plant host. Plant host proteins also play a key role in this infection process (Gelvin, 2000).
Upon integration of the T-DNA into the plant nuclear genome, the enzymes for the biosynthesis of auxins and cytokinins as well as for opine are produced. Studies on this process of Agrobacterium–plant cell interaction had a great impact on plant transformation technology since any DNA of interest can be inserted into the T-DNA, transferred and integrated into a plant cell genome and subsequently expressed in the target plant. So far a broad range of dicotyledonous and some monocotyledonous plants which were considered as recalcitrant species can now be infected by A. tumefaciens.
Recent discoveries have shown that Agrobacterium transformation can be extended to non-plant eukaryotic organisms including fungi, mushrooms and human cells (reviewed by Lacroix et al., 2006).
Brassica rapa is one of the important species in the genus Brassica (Brassicaceae) This group includes plants such as turnip, cauliflower, cabbage, kohlrabi and Brussel sprout which are important vegetables, forage and oilseed crops (Cardoza et al., 2004).
The plants contain a large number of interesting phytochemicals including some with anticancer properties (Christey and Braun, 2004). Considerable progress has been made in recent years to produce transgenic B. rapa that can confer specific desired traits by Agrobacterium-mediated transformation. However, this effort has been hampered partly by inefficient transgene transformation and subsequent regeneration of the targeted plant tissue. Plants infected with pathogens such as Agrobacterium activate a defense signal transduction chain which results in a hypersensitive response (HR) (Kuta and Tripathi, 2005). The HR is thought to be one of the factors that contribute to the inefficiency of the Agrobacterium-mediated transformation system (Gustavo et al., 1998). Previous studies also showed that plant gene expression including defense-related genes was altered in response to A. tumefaciens infection (Ditt et al., 2001; Ditt et al., 2005). Though the effect on transcriptome level has extensively been studied, little information is available on the metabolome level. For further understanding of the plant-Agrobacterium interaction there is a need to investigate the metabolome changes of B. rapa upon infection with A. tumefaciens. The information obtained may also shed new light on how interaction with Agrobacterium will affect the metabolite pool of B. rapa and how that might directly or indirectly influence the transformation efficiency. In the present study we analyzed the metabolome of B. rapa upon infection by disarmed and tumor-inducing strains of A. tumefaciens. 1H-NMR coupled with multivariate data analysis methods, i.e.
PLS-DA and PCA was applied to determine the metabolome changes in B. rapa.
7.2 Materials and methods
7.2.1 Plant materials and growth of Agrobacterium tumefaciens
Four different cultivars of B. rapa; Raapstelen, Herfstraap and Witte Mei, obtained from the Plant Ecology Department, University of Leiden and ssp. oleifera obtained from Plant
Boreal Ltd, Jokioinen, Finland were cultivated from the seeds and grown in a greenhouse (16/8-h (light/dark) photoperiod at 300-500 µmol m-2 s-1). The disarmed Agrobacterium strains LBA4404 carrying the entC gene and the CaMV 35S promoter and the tumor- inducing Agrobacterium strains LBA4001 (octopine) and LBA4902 (nopaline) were used. The bacteria were inoculated in liquid LB medium (1% Bacto tryptone, 0.5%
Bacto yeast extract, 0.5% NaCl) containing the antibiotics rifampicin and kanamycin (50 µg/ml) and grown overnight at 28°C on a rotary shaker at 200 rpm in the dark before inoculation.
7.2.2 Inoculation of Brassica rapa with disarmed strain LBA4404
Inoculation of B. rapa with disarmed Agrobacterium LBA4404 was carried out on six weeks old greenhouse-grown plants. Four varieties, i.e. cv. Raapstelen, Witte Mei, Herfstraap and ssp. oleifera were used in this experiment. The surfaces of the first three lower leaves were wounded with scalpel blades. Subsequently depending on the size of the leaves, 1.0-2.0 ml of bacterial cultures (diluted with distilled water, OD600=1.0-1.5) were sprayed uniformly on both upper and lower sides of leaves. For measuring the systemic effects of infection, the first two upper leaves were analyzed. The control experiments were carried out in the same way but spraying was done with distilled water instead of bacterial cultures. The healthy plants without any wounding were used as blank controls. The leaves were harvested after 7 days of inoculation. The biomass ratio (mg dry weight) of the Agrobacterium (1.0-2.0 ml of cultures) to the individual infected leaves was around 0.3-0.6:4.0-6.0
7.2.3 Inoculation of Brassica rapa with tumor-inducing nopaline (LBA4902) and octopine (LBA4001) strains
To induce tumor growth, the stems of three weeks old greenhouse-grown plants were injected with 1 ml of bacterial cultures (diluted with distilled water, OD600=1.0-1.5) from the tumor-inducing octopine and nopaline strains. Two varieties, i.e. cv. Raapstelen and ssp. oleifera were used in this experiment. The stem below the tumors and the first two upper leaves from tumor-bearing plants were also taken. The tumors as well as the leaves and stems from tumor-bearing plants were harvested when the plants were 8 weeks old.
Three individual plants were used as replicates for each experiment. The control experiments were carried out in the same way but the plants were injected with distilled water instead of bacterial cultures. Healthy plants without any wounding were used as blank controls.
7.2.4 Extraction of tumors and plant materials
The tumors and the plant materials (leaves and stems) were grounded with liquid nitrogen and freeze dried prior to extraction. Fifty milligrams of freeze-dried materials were transferred to a 2 ml Eppendorf tube in which 750 ul of methanol-d4 in D2O and 750 ul of KH2PO4 buffer, pH 6.0 containing 0.1% of trimethyl silyl propionic acid sodium salt (w/v) were added. The mixture was then vortexed for 2 minutes and sonicated for 15 minutes. After sonication, the mixture was spun down at 13,000 rpm for 20 minutes at room temperatures. Eight hundreds microliters of the supernatant were transferred into a 5 mm NMR tube for analysis.
7.2.5 NMR spectra measurement
1H-NMR, 2D-J-resolved spectra were recorded at 25 oC on a 500 MHz Bruker DMX-500 spectrometer (Bruker, Karlsruhe, Germany). 1H-1H-correlated spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bonds coherence (HMBC) spectra were recorded on a 600 MHz Bruker DMX-600 spectrometer (Bruker, Kalsruhe, Germany). All the NMR parameters were the same to those of our previous reports (Jahangir et al., 2008; Abdel-Farid et al., 2007).
7.2.6 Data analysis
Spectral intensities of 1H-NMR spectra were scaled to total intensity and reduced to integrated regions of equal width (0.04) corresponding to the region of δ 0.4- δ 10.0. The regions of δ 4.8-δ 4.9 and δ 3.28-δ 3.40 were excluded from the analysis because of the residual signal of water and MeOH. Principal component analysis (PCA) and partial least square regression analysis (PLS) were performed with the SIMCA-P software (v. 11.0, Umetrics, Umeå, Sweden).
7.3 Results and discussion
7.3.1 Agrobacterium tumefaciens infection
Upon infection with tumor-inducing nopaline or octopine strains, tumors started to form on stems of both varieties of B. rapa (cv. Raapstelen and ssp. oleifera) after one week of infection. Tumors induced by the nopaline strain have a smooth morphology whereas tumors induced by the octopine strain have a rough morphology (Hooykaas et al., 1980) as shown in Figure 7.1. Spraying the 6 weeks old B. rapa plants with disarmed Agrobacterium strain LBA 4404 did not have any obvious visible effect on the treated leaves of all the varieties. Only few leaves produced a slight yellowish colour after 7 days of treatment.
7.3.2 1H-NMR spectral analysis of the metabolites from Brassica rapa infected with disarmed (LBA4404) and tumor-inducing octopine or nopaline strains of Agrobacterium tumefaciens
1H-NMR spectra, 2D spectra (J-resolved, 1H-1H-COSY, HSQC and HMBC), our in- house database of reference compounds and previous reports (Jahangir et al., 2008;
Abdel-Farid et al., 2007) were used for the signal assignment of metabolites from the plants. The list of assigned signals for metabolites is presented in Table 7.1. Compounds that are present in all the samples are alanine, valine, threonine, leucine and isoleucine, succinic acid, fumaric acid, formic acid, glutamic acid and malic acid. Common sugars, glucose and sucrose were also present in all the samples. Arginine and indole acetic acid (IAA) were exclusively detected in the nopaline tumors. Other compounds identified in all the samples are adenine and choline. For secondary metabolites, the signals of progoitrin and the flavonoid quercetin were observed in the nopaline tumors, whereas in the octopine tumors, only quercetin was detected. The signals of phenylpropanoids ((δ 6.48, dd, J=16 Hz), (δ 6.99, s)) were also present in the nopaline and octopine tumors.
Flavonoids (kaempferol and quercetin analog) and phenylpropanoids (cis- and trans- sinapoyl malate and coumaroyl malate) were identified from the leaves infected with strain LBA4404 and the leaves and stems from the tumor-bearing plants. With the aid of J-resolved and 1H-1H-COSY spectra and the octopine reference compound, octopine (δ 1.51 (d, J=8.0, H-3’), δ 3.68 (m, H-2, H-2’), δ 1.9 (m, H-3), δ 1.7 (m, H-4), δ 3.24 (t,
Figure 7.1 Tumor induced in Brassica rapa plant (a) nopaline tumor and (b) octopine tumor on cv. Raapstelen (c) octopine tumor on ssp. oleifera
a
b
c a
c
Table 7.1 The compounds identified from 1H-NMR and J-resolved spectra of octopine and nopaline tumors and disarmed Agrobacterium (LBA4404) infected leaf. (+) : detected, (-) : undetected, s=singlet, d=doublet, dd=double of doublet, t=triplet, td=triple of doublet, m=multiplet.
Compound Chemical shift (δ in ppm) and coupling constant ( J in Hz)
Tumor octopine
Tumor nopaline
LBA4404 infected leaf Amino/organic acid
Alanine δ 1.49 (d, J=7.0), δ 3.76 (m) + + +
Valine δ 1.02 (d, J=8.0), δ 1.06 (d, J=8.0), δ 2.39 ( m)
+ + +
Threonine δ 1.34 (d, J=6.6), δ 4.33 (m) + + +
Leucine δ 0.96 (d, J=6.8), δ 0.98 (d, J=6.8) + + +
Isoleucine δ 0.95 (t, J=7.5 ), δ 1.00 (d, J=7.0), δ 3.65 (d, J=7.0)
+ + +
Arginine δ 3.72 (t, J=8.0 ), δ 3.24 (t, J=8.0), δ 1.7 (m), δ 1.8 (m)
- + -
Glutamic acid δ 2.08 (m), δ 2.14 (m), δ 2.38 (td, J=8.0, 2.0), δ 3.79 (dd, J=7.0, 3.5)
+ + +
Succinic acid δ 2.54 (s) + + +
Fumaric acid δ 6.58 (s) + + +
Formic acid δ 8.49 (s) + + +
Malic acid δ 2.58 (dd, J=17.0, 3.5), δ 2.82 (dd, J=17.0, 3.5) δ 4.32 (dd, J=11.5, 4.0)
+ + +
Indole-3-acetic acid (IAA)
δ 3.23 (d, J=16.0), δ 3.36 (d, J=16.0), δ 7.10 (s), δ 7.13 (t, J=7.8), δ 7.21 (t, J=7.8), δ 7.47 (d, J=8.0), δ 7.72 (dd, J=7.8)
- + -
Sugar
β-glucose δ 4.58 (d, J=8.0, H-1) + + +
α-glucose δ 5.18 (d, J=3.6, H-1)) + + +
Sucrose δ 5.40 (d, J=4.0, H-1) + + +
Fructose moiety of sucrose
δ 4.18 (d, J=8.0) + + +
Phenylpropanoid/
flavonoid/glucosinolate
Kaempferol analogue δ 7.96 (d, J=8.8), δ 6.82 (d, J=2.0), δ 7.00 (d, J=8.8), δ 6.45 (d, J=2.0)
- - +
Quercetin analogue δ 6.49 (d, J=2.2), δ 6.76 (d, J=2.2), δ 6.92 (d, J=9.2) δ 7.71 (d, J=9.2), δ 7.79 (d, J=2.2)
+ + +
trans - sinapoyl malate δ 7.65 (d, J=16.0), δ 6.49 (d, J=16.0), δ 6.97 (s), δ 3.88 (s)
- - +
cis - sinapoyl malate δ 6.95 (d, J=13.0), δ 5.95 (d, J=13.0), δ 7.11 (s)
- - +
trans - coumaroyl malate
δ 7.66 (d, J=16.0), δ 7.57 (d, J=8.8), δ 6.85 (d, J=8.8), δ 6.46 (d, J=16.0)
- - +
cis - coumaroyl malate δ 7.92 (d, J=10.0), δ 6.96 (d, J=10.0), δ 7.02 (d, J=10.0)
- - +
Progoitrin δ 5.96 (m), δ 4.64 (m), δ 5.34 (td, J=10.0, 3.0), δ 5.21 (td, J=10.0, 3.0), δ 2.95, (dd, J=17.0, 3.0), δ 2.92 (dd, J=17.0, 9.0)
- + -
Other compound
Octopine δ 1.51 (d, J=8.0), δ 3.68 (m), δ 1.7 (m), δ 1.9 (m), δ 3.24 (t, J=8.0)
+ - -
Adenine δ 8.20 (s), δ 8.21(s) + + +
Choline δ 3.22 (s) + + +
Figure 7.2 Typical 1H-NMR spectra of (a) nopaline tumor (b) octopine tumor (c) disarmed Agrobacterium (LBA4404) infected leaf. Assigned signals: 1. IAA 2. fumaric acid 3. progoitrin 4. octopine 5. alanine 6. threonine 7. valine 8. leucine/isoleucine 9.
sinapoyl malate 10. coumaroyl malate
a
b
c
7 8
6 4 5
2 3 1
9
10 9 10 9
J=8.0, H-5) was identified in the octopine tumors. However, the signal of nopaline could not be detected in the samples of tumors induced by the nopaline strain. The typical 1H- NMR spectra from nopaline and octopine tumors as well as the leaves infected with the disarmed strain LBA4404 are shown in Figure 7. 2.
7.3.3 Multivariate analysis of the 1H-NMR spectra
7.3.3.1 Effect of the disarmed (LBA4404) strain of Agrobacterium tumefaciens on Brassica rapa metabolites
Figure 7.3a-c shows the PLS-DA score and loading plots of B. rapa leaves infected with disarmed strain (LBA4404) of A. tumefaciens. In the score plot (PLS component 1 vs.
PLS component 2) (Figure 7.3a), a distinct separation was found between the local infected leaves and their controls. However there was no clear separation between the systemic leaves and their controls. The corresponding loading scatter plot (Figure 7.3b) reveals that the signals with the highest impact in local infected leaves are all amino acids (alanine, threonine, valine and glutamic acid) whereas the signals that gave the highest impact for control plants were flavonoids (quercetin and kaempferol analogs), phenylpropanoids (trans- and cis- sinapoyl malate and coumaroyl malate) as well as formic acid. The loading column plot (Figure 7.3c) clearly shows that the negative side of PLS component 2 which corresponds to local infected leaves is mostly dominated by the signals of compounds in the aliphatic region including amino acids, whereas the positive side of PLS component 2 which corresponds to the control leaves is dominated by the signals of compounds in the aromatic region including secondary metabolites, flavonoids and phenylpropanoids. The metabolites identified in systemic leaves and their controls were all organic acids i.e. succinic acid, fumaric acid, malic acid as well as sucrose and glucose. These results suggest that the infection of B. rapa leaves with disarmed strains of A. tumefaciens suppressed the accumulation of these flavonoids and phenylpropanoids, the compounds that usually are induced in the plants in response to biotic and abiotic stress. In contrast to this, the systemic infection of the leaves did not cause any alteration of flavonoids and phenylpropanoids in comparison to the controls.
Principal component analysis (PCA) was also performed to investigate the effect of mechanically wounded leaves against the healthy leaves (blank control). PCA showed
-10 0 10
-20 -10 0 10 20
1
1 1
1
1 1
1
1 1
1
1 1 2
2 2
2 2 2
2
2 2
2 2 2
3 3
3
3 3
3 3 3 3 3 3
3 4
4 4
4 4 4
4 4
4
4
4 4
-0.1 0.0 0.1
-0.10 -0.08 -0.06 -0.04 -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12
-0.1 0.0 0.1
8.56 8.4 8.2 8 7.8 7.6 7.4 7.2 7 6.8 6.6 6.4 6.2 6 5.8 5.6 5.4 5.2 5 4.56 4.36 4.16 3.96 3.76 3.56 3.36 3.12 2.92 2.72 2.52 2.32 2.12 1.92 1.72 1.52 1.32 1.12 0.92 0.72
Figure 7.3 The score plot (a) and loading scatter plot (b) of Brassica rapa leaves infected with disarmed strain (LBA4404) of Agrobacterium tumefaciens 1. Local infected leaves 2. Control for local infected leaves 3. Systemic leaves 4. Control for systemic leaves. Loading column plot (c) showing the metabolites responsible for the separation along the PC2. Assigned signals: 1. formic acid 2. sinapoyl malate 3. coumaroyl malate 4. kaempferol 5. quercetin 6. glutamic acid 7. alanine 8. threonine 9.
valine
1
wc*[1]
wc*[2] PLS component 2
4
9 PLS component 1 (23%)
PLS component 2 (19%)
2
3 4
succinic acid, fumaric acid, malic acid, sucrose, glucose
alanine, threonine, valine, glutamic acid
kaempferol, quercetin, sinapoyl malate, coumaroyl malate formic acid,
2 3 5
4
6
7 8
b
c
1
control for local infected leaves
local infected leaves systemic leaves + control
for systemic leaves
a
that there was induction of flavonoids and phenylpropanoids compound as well as amino and organic acids in comparison to the healthy leaves (blank controls) (data not shown).
7.3.3.2 Effect of tumor-inducing nopaline and octopine strains on Brassica rapa metabolites
The score plot (PLS component 2 vs. PLS component 3) of PLS-DA (Figure 7.4a) shows a clear discrimination among the different groups of samples. Samples of tumors, leaves/stems from tumor-bearing plants, leaves/stems from control plants (injected with only distilled water) as well as leaves/stems from blank control plants were clearly separated from each other. The corresponding signals in the loading plot (Figure 7.4b) suggest that the tumors induced by the nopaline and octopine strains have higher concentrations of succinic acid and glutamic acid. The leaves/stems of tumor-bearing plants show higher concentration of alanine, threonine, leucine/isoleucine, formic acid and adenine. In contrast to this, the leaves/stems from control plants were found to have higher concentrations of fumaric acid, malic acid, kaempferol and a quercetin analog as well as cis- and trans- sinapoyl malate and coumaryl malate. This result shows that the presence of tumors in the infected plants might suppress the accumulation of certain flavonoids and phenylpropanoids in the leaves/stems taken from the tumor-bearing plants of B. rapa. The leaves/stems from healthy plants without any wounding which serve as blank controls have higher concentrations of sugars, glucose and sucrose in comparison with the leaves/stems from tumor-bearing plants and control plants.
Principal component analysis (PCA) was performed to identify the distinctive metabolites present in nopaline and octopine tumors. The PCA score plot (PC2 vs. PC3) (Figure 7.5a) clearly shows the separation between these two tumors. The corresponding signals in the loading column plot along the PC3 (Figure 7.5b) suggest that tumors induced by the octopine strain (located in the negative side of PC3) have higher concentrations of amino acids (threonine, valine, leucine/isoleucine), organic acids (succinic acid, malic acid, formic acid, fumaric acid), adenine, sucrose and the flavonoid quercetin, whereas the tumors induced by the nopaline strain (located in the positive side of PC3) have higher concentrations of alanine, glutamic acid, glucose and phenylpropanoid derivatives.
-8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8
-10 0 10
1 1
11 1
1 1 1 1
1
1 2
2 2
2 2
2 2
2 2
2 2
333 3
3
3
33
3
3 3
4
4 4 4
4 4
4 4 4 4 4 2 2
2
2 2 2
2 2 2
2
22 3
4
-0.2 -0.1 -0.0 0.1 0.2
-0.1 0.0 0.1
Figure 7.4 The score plot (a) and loading scatter plot (b) of Brassica rapa infected with tumor-inducing octopine and nopaline strains of Agrobacterium tumefaciens 1. Tumors induced by octopine/nopaline strains 2. Leaves/stems from tumor–bearing plants 3.
Leaves/stems from blank control plants 4. Leaves/stems from control plants (injected with distilled water).
As previously mentioned, IAA, arginine and progoitrin were found in the tumors induced by the nopaline strain. Crown gall tumors are reported to contain high levels of auxins and cytokinins synthesized by the enzymes encoded by the T-DNA that is integrated into the genome of host plant (Weiler and Kurt, 1981). Tumor growth is thought to be dependent on overproduction of auxins (Schwalm et al., 2003) and these compounds together with cytokinins, ethylene and abscisic acid are involved in proliferation and vascularization of tumor tissue (Veselov et al., 2003) to promote
4
wc*[2]
wc* [3]
1
2
3
kaempferol, quercetin sinapoyl malate coumaroyl malate fumaric acid, malic acid succinic acid,
glutamic acid
alanine, leucine/isoleucine threonine, formic acid,
adenine glucose, sucrose
b
PLS component 2 (27%)
a
leaves/stems from blank control plants
leaves/stems from control plants leaves/stems from
tumor-bearing plants
octopine/nopaline tumors
PLS component 3 (18%)
-10 0 10
-20 -10 0 10 20
-0.1 0.0 0.1
8.6 8.48 8.32 8.16 8 7.84 7.68 7.52 7.36 7.2 7.04 6.88 6.72 6.56 6.4 6.24 6.08 5.92 5.76 5.6 5.44 5.28 5.12 4.72 4.56 4.4 4.24 4.08 3.92 3.76 3.6 3.44 3.24 3.08 2.92 2.76 2.6 2.44 2.28 2.12 1.96 1.8 1.64 1.48 1.32 1.16 1 0.84 0.68
Figure 7.5 (a) The score plot of PCA of nopaline and octopine tumors induced in Brassica rapa after infection with tumor-inducing nopaline and octopine strains of Agrobacterium tumefaciens. (b) The loading column plot of PCA along the PC3.
Assigned signal: 1 formic acid. 2. adenine 3. quercetin 4. fumaric acid 5.
phenylpropanoid 6. sucrose 7. glucose 8. malic acid 9. succinic acid 10. glutamic acid 11. alanine 12. threonine 13. valine 14. leucine/isoleucine
efficient supply of nutrients and water. Liu and Nester, (2006) suggested that IAA not only acts as inhibitor for the vir gene expression when the expression is no longer needed but it might also function as a metabolite that can serve as a chemical agent in plant defense against a wide variety of plant associated bacteria in the rhizosphere. As a nopaline tumor is formed by the direct condensation of arginine and α-ketoglutaric acid, whereas an octopine tumor results from condensation of arginine and pyruvic acid (Christou et al., 1985), arginine is expected to be present in the crown gall tumors.
PC 2 (41%)
PC 3 (22%) PC 3
octopine tumors
nopaline tumors
a
b
3 9
5 10
11
8 14
12 2 7
1
4
2 6
13
However, in our case, arginine was only found in nopaline tumors whereas α-ketoglutaric acid and pyruvic acid were not detected in the respective tumors.
This study also shows the presence of the flavonoid quercetin and phenylpropanoids in nopaline and octopine tumors. Previous work on A. tumefaciens on white clover (Trifolium repens (L)) with the β-glucuronidase (GUS)-fused auxin-responsive promoter (Schwan et al., 2003) showed that the plant tumors induced by A. tumefaciens accumulated considerable amounts of kaempferol as well as flavonoid aglycone i.e. 7,4 dihydroflavone (DHF), formononetin and medicarpin. However none of these flavonoids were detected in our study.
Plants exposed to stress such as being infected by pathogens or caused by mechanical wounding show many distinct biochemical changes including the metabolites originating from phenylpropanoid pathways (Richard et al., 2000; Dixon and Paiva, 1995). These compounds are produced at the site of infection or systemically in unwounded tissue (Richard et al., 2000; Ryan, 1990). The carbon flow is increased from primary metabolism towards phenylpropanoid metabolism which leads, among others, to the biosynthesis of phenylpropanoid-derived compounds (Ellard-Ivey and Douglas, 1996).
The induction of certain flavonoids and phenylpropanoids and the fluctuation of primary metabolites (sugars and amino/organic acids) in the plants caused by mechanical wounding as well as in the nopaline and octopine tumors as shown by our results are in line with this. In the past years, many studies have demonstrated the role of hydroxycinnamic acid derivatives in plant defense responses to biotic or abiotic stress (Daayf et al., 2000; Janas et al., 2000). Housti et al. (2002) reported the accumulation of caffeoyl malate, feruloyl malate and p-coumaroyl malate in the leaves of Thunbergia alata when these plants were treated with salicylic acid and upon wounding locally or systemically. In contrast, our result from the leaves infected with disarmed strain of A.
tumefaciens suggests the suppression of flavonoids and phenylpropanoids. The leaves and stems taken from tumor-bearing plants infected with octopine and nopaline strains also showed a similar phenomenon. Similar results were reported by Hagermeier et al.
(2001) who observed the absence of phenylpropanoid compounds when Arabidopsis thaliana was infected with Pseudomonas syringae. Tan et al. (2001), by using the same
species of Arabidopsis and pathogen, observed a decrease of prominent phenylpropanoid metabolites, but several cell wall-bound phenylpropanoids increased at the same time.
7.4 Conclusion
It was shown by this study that infection of B. rapa leaves with disarmed strain of A.
tumefaciens resulted in suppression of certain flavonoids and phenylpropanoids. The similar phenomenon was also observed from the leaves and stems taken from tumor- bearing plants infected with octopine and nopaline strains. The results also showed that nopaline and octopine tumors differ in the concentrations of certain metabolites between each other.
7.5 Acknowledgements
The authors thank Prof. P.J.J. Hooykaas for providing the different strains of Agrobacterium. We also thank Malaysian Agricultural Research and Development Institute (MARDI), Malaysia for the Ph.D grant to Sanimah Simoh.
