Professional Documents
Culture Documents
DOI 10.1007/s11306-016-1037-2
ORIGINAL ARTICLE
Abstract
Introduction Metabolomics provides a view of endogenous metabolic patterns not only during plant growth,
development and senescence but also in response to genetic
events, environment and disease. The effects of the field
environment on plant hormone-specific metabolite profiles
are largely unknown. Few studies have analyzed useful
phenotypes generated by introducing single or multiple
gene events alongside the non-engineered wild type control
at field scale to determine the robustness of the genetic trait
123
103
Page 2 of 15
1 Introduction
Metabolomics provides a view of endogenous metabolic
patterns not only during plant growth, development and
senescence but also in response to genetic events, biodiversity, environment and disease. Thus, to mention a few
examples, metabolomics has contributed to mapping plant
cell types (Moussaieff et al. 2013), modeling plant
123
T. Fatima et al.
Fruit metabolite networks in engineered and non-engineered tomato genotypes reveal fluidity
Page 3 of 15
103
123
103
Page 4 of 15
123
T. Fatima et al.
Fruit metabolite networks in engineered and non-engineered tomato genotypes reveal fluidity
Page 5 of 15
103
Univariate analyses were performed with analysis of variance (ANOVA) using the mixed model procedure of SAS
version 9.2 (SAS Institute, Cary, NC). The model for
analyzing fruit yield variables, photosynthesis variables,
polyamines and metabolites had mulch, genotype and
mulch by genotype interaction treatments as fixed effects.
Year and field block nested within year were random
effects for the fruit yield analyses, year and block within
year and sampling date within year were random effects for
the photosynthesis analyses, and year and fruit stage were
random effects for the polyamine and metabolite analyses.
Principal components analysis (PCA) was performed on
the metabolite data set after standardization to mean zero
and standard deviation one. Two ANOVAs were performed, one for each of the first two components using
mulch, genotype and mulch by genotype interaction treatments as fixed effects.
Metabolites were organized into groupings of amino
acids, organic acids, sugars, and energy metabolites in
order to facilitate analysis of overall trends within groups.
Each metabolite was standardized to mean = 0 and standard deviation = 1 to give equal weighting to each
metabolite. An ANOVA was then performed on the standardized metabolic data by group with genotype or mulch
treatment as a fixed effect.
Canonical correlation analysis (CCA) was performed to
explore multivariate correlations between whole plant
variables (early fruit number, early fruit weight, early
weight per fruit, total fruit number, total fruit weight, total
weight per fruit, net photosynthesis, stomatal conductance,
and internal CO2 concentration) and metabolite variables.
Univariate Pearson correlations between these variables
were also calculated.
A single network analysis for metabolites (Langfelder
and Horvath 2008; DiLeo et al. 2011), assuming an
unvarying correlation structure among variables over the
whole data set, was found not adequate to describe our
metabolite networks and their dynamics under the
123
103
Page 6 of 15
123
T. Fatima et al.
3 Results
3.1 Confirmation of system function under field
grown conditions
We first ascertained the stability of the introduced genes in
tomato plants in the field and/or their biochemical phenotype. In addition to the engineered lines mentioned above,
we developed and tested additional tomato lines with
double genetic events by making backcrosses between one
high polyamine line (line 10) and ethylene-suppressed line
2 (102AS-1HO, line 4), as well as another high polyamine
line (line 8) and meJAS-deficient line 12 (LS-4HO, line 20)
with an azygous line as a control (description in Supplementary Table 1). Q-PCR data of three genesyeast SAM
decarboxylase (ySAMDC), tomato E8 (SlE8) and tomato
lipoxygenase (SlLOX) in different tomato genotypes grown
in BP (black plastic mulch) and HV (hairy vetch mulch) are
shown in Supplementary Fig. 1. As expected, lines 8 and
10, carrying the ySAMDC transgene, expressed ySAMDC
transcripts. However, line 20 had higher expression of
ySAMDC transcripts than its parent line 8. Specific effects
of the BP versus HV were evident: ySAMDC transcript
expression in line 20 (G20) was several-fold enhanced
under BP (Supplementary Fig. 1, ySAMDC). To ascertain
if this BP effect was due to an effect on the SlE8 promoter
used to drive the expression of the chimeric ySAMDC
construct, the accumulation of SlE8 transcripts was also
quantified under BP and HV. The pattern of SlE8 transcript
accumulation in BP-grown lines 8 and 10 was similar to the
pattern of ySAMDC transcripts (Supplemental Fig. 1,
compare E8 with ySAMDC under BP). In fact, BP-grown
line 12 had higher expression of SlE8 transcripts than when
grown under HV (Supplementary Fig. 1, E8), and in contrast these transcripts in control azygous line 5 accumulated
maximally under HV. SlLOX transcripts were suppressed
in both line 12 and its sibling, genotype 20, as expected
while mulch systems had little differential effect. HV
mulch had a more enhancing effect on SlLOX expression in
line 5 (G5) and line 8 (G8). In general, the relative accumulation levels of SlE8 transcripts were considerably lower
than ySAMDC and SlLOX transcripts.
We introduced SAMDC into tomato (Mehta et al. 2002)
because we anticipated that among the main polyamines
present in tomatoputrescine (Put), spermidine (Spd) and
spermine (Spm)Spd and Spm should accumulate and Put
should decline in lines 8 and 10, and their siblings 4 and 20.
The values for the three polyamines (Supplementary
Table 4) showed the expected pattern of polyamines for the
indicated genotypes. The siblings (lines 4 and 20) behaved
like their high polyamine parents qualitatively and quantitatively, with line 20 having elevated spermine levels
Fruit metabolite networks in engineered and non-engineered tomato genotypes reveal fluidity
Page 7 of 15
103
123
103
Page 8 of 15
T. Fatima et al.
123
Fruit metabolite networks in engineered and non-engineered tomato genotypes reveal fluidity
Page 9 of 15
103
123
103
Page 10 of 15
T. Fatima et al.
are the darkest red, the most stable are yellow. The upper right
triangle gives the average correlation of each pair, the lower left
triangle gives the standard deviations used to create the heat map
123
Fruit metabolite networks in engineered and non-engineered tomato genotypes reveal fluidity
Page 11 of 15
103
123
103
Page 12 of 15
T. Fatima et al.
indicating the magnitude of change. Note that there are more changes
for low than high polyamine content genotypes, suggesting that
effects of mulch are more pronounced for low polyamine content
genotypes; see text for further explanation
4 Discussion
123
Fruit metabolite networks in engineered and non-engineered tomato genotypes reveal fluidity
Page 13 of 15
103
5 Conclusions
This study portrays how metabolite relationships change
under different genotypic and environmental conditions.
Although these networks are surprisingly dynamic,
123
103
Page 14 of 15
examples of selectively conserved associations were evident. The importance of the specific agroecosystem environment in metabolomics research is highlighted;
metabolic profiles are highly sensitive to the nature of the
growing conditions under which tomato plants are grown
and fruit is ripened. The development of transgenic
germplasm will be subject to the same genotype by environment interactions that traditional breeders have
addressed for centuries. Future research is needed to
identify other specific traits that modulate metabolomic
profiles in fruit and better define how expressions of these
traits are modified by agroecosystem environment.
Acknowledgments We thank numerous students and postdoctoral
associates for their help during harvesting/processing of the fruits, and
Tatsiana Datsenka for assistance in developing the methyl jasmonate
deficient X high polyamine double transgenic genotype. Mention of
trade names or commercial products is solely for the purpose of
providing specific information and does not imply recommendation or
endorsement by the U.S. Department of Agriculture (USDA). USDA
is an Equal Opportunity Provider.
Compliance with ethical standards
Conflict of interest None.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
References
Abdul-Baki, A. A., & Teasdale, J. R. (2007). Sustainable production
of fresh-market tomatoes and other vegetables with cover crop
mulches. USDA-ARS Farmers Bulletin No. 2280. Beltsville,
MD.
Abdul-Baki, A. A., Teasdale, J. R., Korcak, R. F., Chitwood, D. J., &
Huettel, R. N. (1996). Freshmarket tomato production in a lowinput alternative system using cover-crop mulch. Journal of the
American Society for Horticultural Science, 31, 6569.
Aizat, W. M., Dias, D. A., Stangoulis, J. C. R., Able, J. A., Roessner,
U., & Able, A. J. (2014). Metabolomics of capsicum ripening
reveals modification of the ethylene related-pathway and carbon
metabolism. Postharvest Biology and Technology, 89, 1931.
Alcazar, R., Altabella, T., Marco, F., Bortolotti, C., Reymond, M.,
Koncz, C., et al. (2010). Polyamines: Molecules with regulatory
functions in plant abiotic stress tolerance. Planta, 231,
12371249.
Benjamini, Y., & Hochberg, Y. (1995). Controlling the false
discovery rate: A practical and powerful approach to multiple
testing. Journal of the Royal Statistical Society. Series B, 57,
289300.
Brady, S. M., Benfey, P. N., & Aharoni, A. (2013). High-resolution
metabolic mapping of cell types in plant roots. Proceedings of
the National Academy of Sciences, 110, E1232E1241.
123
T. Fatima et al.
Buyer, J. S., Teasdale, J. R., Roberts, D. P., Zasada, I. A., & Maul, J.
E. (2010). Factors affecting soil microbial community structure
in tomato cropping systems. Soil Biology & Biochemistry, 42,
831841.
Capell, T., Bassie, L., & Christou, P. (2004). Modulation of the
polyamine biosynthetic pathway in transgenic rice confers
tolerance to drought stress. Proceedings of the National
Academy of Sciences of the United States of America, 101,
99099914.
Carrari, F., Baxter, C., Usadel, B., Urbanczyk-Wochniak, E., Zanor,
M.-I., Nunes-Nesi, A., et al. (2006). Integrated analysis of
metabolite and transcript levels reveals the metabolic shifts that
underlie tomato fruit development and highlight regulatory
aspects of metabolic network behavior. Plant Physiology, 142,
13801396.
Centeno, D. C., Osorio, S., Nunes-Nesi, A., Bertolo, A. L., Carneiro,
R. T., Araujo, W. L., et al. (2011). Malate plays a crucial role in
starch metabolism, ripening, and soluble solid content of tomato
fruit and affects postharvest softening. Plant Cell., 23, 162184.
Cohen, S., Chang, A., Boyer, H., & Helling, R. (1973). Construction
of biologically functional bacterial plasmids in vitro. Proceedings of the National Academy of Sciences of the United States of
America, 70, 32403244.
Colombie, S., Nazaret, C., Benard, C., Biais, B., Mengin, V., Sol, M.,
et al. (2015). Modelling central metabolic fluxes by constraintbased optimization reveals metabolic reprogramming of developing Solanum lycopersicum (tomato) fruit. The Plant Journal,
81, 2439.
Cramer, G. R., Urano, K., Delrot, S., Pezzotti, M., & Shinozaki, K.
(2011). Effects of abiotic stress on plants: A systems biology
perspective. BMC Plant Biology, 11, 163.
DiLeo, M. V., Strahan, G. D., den Bakker, M., & Hoekenga, O. A.
(2011). Weighted correlation network analysis (WGCNA)
applied to the tomato fruit metabolome. PLoS One, 6(10),
e26683.
Dobritzsch, S., Weyhe, M., Schubert, R., Dindas, J., Hause, G.,
Kopka, J., et al. (2015). Dissection of jasmonate functions in
tomato stamen development by transcriptome and metabolome
analyses. BMC Biology, 13, 28.
Fatima, T., Teasdale, J. R., Bunce, J., & Mattoo, A. K. (2012).
Tomato response to legume cover crop and nitrogen: Differing
enhancement patterns of fruit yield, photosynthesis and gene
expression. Functional Plant Biology, 39, 246254.
Flores, H. E., & Filner, P. (1985). Polyamine catabolism in higher
plants: Characterization of pyrroline dehydrogenase. Plant
Growth Regulation, 3, 277291.
Fukushima, A., Kusano, M., Redestig, H., Arita, M., & Saito, K.
(2011). Metabolomic correlation-network modules in Arabidopsis based on a graph-clustering approach. BMC Systems Biology,
5, 1.
Grokinsky, D. K., Svensgaard, J., Christensen, S., & Roitsch, T.
(2015). Plant phenomics and the need for physiological phenotyping across scales to narrow the genotype-to-phenotype
knowledge gap. Journal of Experimental Botany,. doi:10.1093/
jxb/erv345.
Hall, R. D., Brouwer, I. D., & Fitzgerald, M. A. (2008). Plant
metabolomics and its potential application for human nutrition.
Physiologia Plantarum, 132, 162175.
Kausch, K. D., Sobolev, A. P., Goyal, R. K., Fatima, T., Laila-Beevi,
R., Saftner, R. A., & Mattoo, A. K. (2012). Methyl jasmonate
deficiency alters cellular metabolome, including the aminome of
tomato (Solanum lycopersicum L.) fruit. Amino Acids, 42,
843856.
Kogel, K.-H., Voll, L. M., Schafer, P., Jansen, C., Wu, Y., Langen,
G., et al. (2010). Transcriptome and metabolome profiling of
field-grown transgenic barley lack induced differences but show
Fruit metabolite networks in engineered and non-engineered tomato genotypes reveal fluidity
cultivar-specific variances. Proceedings of the National Academy of Sciences of the United States of America, 107,
61986203.
Kugler, P., & Yang, W. (2014). Identification of alterations in the
Jacobian of biochemical reaction networks from steady state
covariance data at two conditions. Journal of Mathematical
Biology, 68, 17571783.
Kumar, V., Mills, D. J., Anderson, J. D., & Mattoo, A. K. (2004). An
alternative agriculture system is defined by a distinct expression
profile of select gene transcripts and proteins. Proceedings of the
National Academy of Sciences of the United States of America,
101, 1053510540.
Langfelder, P., & Horvath, S. (2008). WGCNA: An R package for
weighted correlation network analysis. BMC Bioinform., 9, 559.
Lee, J. M., Joung, J.-G., McQuinn, R., Chung, M.-Y., Fei, Z., Tieman,
D., et al. (2012). Combined transcriptome, genetic diversity and
metabolite profiling in tomato fruit reveals that the ethylene
response factor SlERF6 plays an important role in ripening and
carotenoid accumulation. The Plant Journal, 70, 191204.
Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene
expression data using real-time quantitative PCR and the
2(DDCT) method. Methods, 25, 402408.
Lu, Y. C., Watkins, K. B., Teasdale, J. R., & Abdul-Baki, A. A.
(2000). Cover crops in sustainable food production. Food
Reviews International, 16, 121157.
Mattoo, A. K. (2014). Translational research in agricultural biology
enhancing crop resistivity against environmental stress alongside
nutritional quality. Frontiers in Chemistry, 1, 30.
Mattoo, A. K., Sobolev, A. P., Neelam, A., Goyal, R. K., Handa, A.
K., & Segre, A. L. (2006). NMR spectroscopy-based metabolite
profiling of transgenic tomato fruit engineered to accumulate
spermidine and spermine reveals enhanced anabolic and nitrogen-carbon interactions. Plant Physiology, 142, 17591770.
Mattoo, A. K., & Teasdale, J. R. (2010). Ecological and genetic
systems underlying sustainable horticulture. Horticultural
Reviews, 37, 331362.
Mattoo, A. K., Upadhyay, R. K., & Rudrabhatla, S. (2015). Abiotic
stress in crops: Candidate genes, osmolytes, polyamines, and
biotechnological intervention. In G. K. Pandey (Ed.), Elucidation of abiotic stress signaling in plants (Vol. 2, pp. 415438).
New York: Springer.
Mehta, R. A., Cassol, T., Li, N., Ali, N., Handa, A. K., & Mattoo, A.
K. (2002). Engineered polyamine accumulation in tomato
enhances phytonutrient content, juice quality and vine life.
Nature Biotechnology, 20, 613618.
Michaeli, S., & Fromm, H. (2015). Closing the loop on the GABA
shunt in plants: Are GABA metabolism and signalling entwined?
Frontiers in Plant Science, 6, 419.
Minocha, S. C., Minocha, R., & Robie, C. A. (1990). Highperformance liquid chromatographic method for the determination of dansylpolyamines. Journal of Chromatography, 511,
177183.
Moussaieff, A., Rogacheva, I., Brodsky, L., Malitskya, S., Toalc, T.
W., Belcherd, H., et al. (2013). High-resolution metabolic
mapping of cell types in plant roots. Proceedings of the National
Academy of Sciences of the United States of America, 110,
E1232E1241.
Neelam, A., Cassol, T., Mehta, R. A., Abdul-Baki, A. A., Sobolev, A.,
Goyal, R. K., et al. (2008). A field-grown transgenic tomato line
expressing higher levels of polyamines reveals legume cover
crop mulch-specific perturbations in fruit phenotype at the levels
Page 15 of 15
103
of metabolite profiles, gene expression and agronomic characteristics. Journal of Experimental Botany, 59, 23372346.
Omranian, N., Kleessen, S., Tohge, T., Klie, S., Basler, G., MuellerRoeber, B., et al. (2015). Differential metabolic and coexpression networks of plant metabolism. Trends in Plant Science, 20,
266268.
Patterson, B. D., & Graham, D. (1987). Temperature and metabolism.
In D. D. Davies (Ed.), The biochemistry of plants (Vol. 12,
pp. 153199). New York: Academic Press.
Ratcliffe, R. G., & Shachar-Hill, Y. (2006). Measuring multiple fluxes
through plant metabolic networks. Plant J., 45, 490511.
Revelle, W. (2014). psych: Procedures for personality and psychological research. Evanston, Illinois: Northwestern University.
http://CRAN.R-project.org/package=psych Version = 1.4.8.
Shannon, P., Markiel, A., Ozier, O., Baliga, N. S., Wang, J. T.,
Ramage, D., et al. (2003). Cytoscape: A software environment
for integrated models of biomolecular interaction networks.
Genome Research, 13, 24982504.
Shelp, B. J., Bown, A. W., & McLean, M. D. (1999). Metabolism and
functions of gamma-aminobutyric acid. Trends in Plant Science,.
doi:10.1111/nph.12283.
Shelp, B. J., Bozzo, G. G., Trobacher, C. P., Zarie, A., Deyman, K. L.,
& Brikis, C. J. (2012). Hypothesis/review: Contribution of
putrescine to 4-aminobutyrate (GABA) production in response to
abiotic stress. Plant Science, 193194, 130135.
Sobolev, A. P., Neelam, A., Fatima, T., Shukla, V., Handa, A. K., &
Mattoo, A. K. (2014). Genetic introgression of ethylenesuppressed transgenic tomatoes with higher-polyamines trait
overcomes many unintended effects due to reduced ethylene on
the primary metabolome. Frontiers in Plant Science, 5, 632.
Sobolev, A. L., Segre, A. L., & Lamanna, R. (2003). Proton high-field
NMR study of tomato juice. Magnetic Resonance in Chemistry,
41, 237245.
Steuer, R., Kurths, J., Fiehn, O., & Weckwerth, W. (2003). Observing
and interpreting correlations in metabolomic networks. Bioinformatics, 19, 10191026.
Takayama, M., & Ezura, H. (2015). How and why does tomato
accumulate a large amount of GABA in the fruit? Frontiers in
Plant Science, 6, 612.
R Core Team (2014). R: A language and environment for statistical
computing. Vienna, Austria: R Foundation for Statistical Computing. URL http://www.R-project.org.
Teasdale, J. R., & Abdul-Baki, A. A. (1997). Growth analysis of
tomatoes in black polyethylene and hairy vetch production
systems. HortScience, 32, 659663.
Toubiana, D., Semel, Y., Tohge, T., Beleggia, R., Cattivelli, L.,
Rosental, L., et al. (2012). Metabolic profiling of a mapping
population exposes new insights in the regulation of seed
metabolism and seed, fruit, and plant relations. PLoS Genetics,
8(3), e1002612.
Vandewalle, I., & Olsson, R. (1983). The gamma-aminobutyric acid
shunt in germinating Sinapis alba seeds. Plant Science Letters,
31, 269273.
Yang, R., Guo, Q., & Gu, Z. (2013). GABA shunt and polyamine
degradation pathway on g-aminobutyric acid accumulation in
germinating fava bean (Vicia faba L.) under hypoxia. Food
Chemistry, 136, 152159.
Zicker, S. C., Wedekind, K. J., & Jewell, D. E. (2006). Antioxidants
in veterinary nutrition. Veterinary Clinics of North America:
Small Animal Practice, 36, 11831198.
123