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. 2013 Aug;75(3):417-429.
doi: 10.1111/tpj.12210. Epub 2013 May 17.

Quantification of growth-defense trade-offs in a common currency: nitrogen required for phenolamide biosynthesis is not derived from ribulose-1,5-bisphosphate carboxylase/oxygenase turnover

Lynn Ullmann-Zeunert #  1   2 , Mariana A Stanton #  1 , Nathalie Wielsch  3 , Stefan Bartram  4 , Christian Hummert  5 , Aleš Svatoš  3 , Ian T Baldwin  1 , Karin Groten  1
Affiliations

Quantification of growth-defense trade-offs in a common currency: nitrogen required for phenolamide biosynthesis is not derived from ribulose-1,5-bisphosphate carboxylase/oxygenase turnover

Lynn Ullmann-Zeunert et al. Plant J. 2013 Aug.

Abstract

Induced defenses are thought to be economical: growth and fitness-limiting resources are only invested into defenses when needed. To date, this putative growth-defense trade-off has not been quantified in a common currency at the level of individual compounds. Here, a quantification method for ¹⁵N-labeled proteins enabled a direct comparison of nitrogen (N) allocation to proteins, specifically, ribulose-1,5-bisposphate carboxylase/oxygenase (RuBisCO), as proxy for growth, with that to small N-containing defense metabolites (nicotine and phenolamides), as proxies for defense after herbivory. After repeated simulated herbivory, total N decreased in the shoots of wild-type (WT) Nicotiana attenuata plants, but not in two transgenic lines impaired in jasmonate defense signaling (irLOX3) and phenolamide biosynthesis (irMYB8). N was reallocated among different compounds within elicited rosette leaves: in the WT, a strong decrease in total soluble protein (TSP) and RuBisCO was accompanied by an increase in defense metabolites, irLOX3 showed a similar, albeit attenuated, pattern, whereas irMYB8 rosette leaves were the least responsive to elicitation, with overall higher levels of RuBisCO. Induced defenses were higher in the older compared with the younger rosette leaves, supporting the hypothesis that tissue developmental stage influences defense investments. We propose that MYB8, probably by regulating the production of phenolamides, indirectly mediates protein pool sizes after herbivory. Although the decrease in absolute N invested in TSP and RuBisCO elicited by simulated herbivory was much larger than the N-requirements of nicotine and phenolamide biosynthesis, ¹⁵N flux studies revealed that N for phenolamide synthesis originates from recently assimilated N, rather than from RuBisCO turnover.

Keywords: Manduca sexta; Nicotiana attenuata; R2R3-MYB transcription factor; caffeoyl-putrescine; dicaffeoyl-spermidine; nicotine; ribulose-1,5-bisphosphate carboxylase/oxygenase; total soluble protein.

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Figures

Figure 1
Figure 1
Overview of experimental strategy used to study growth-defense trade-offs in Nicotiana attenuata in a common nitrogen (N) currency. a) The biosynthesis of nicotine, caffeoyl-putrescine (CP) and dicaffeoyl-spermidine (DCS) is induced after simulated herbivory in wild type (WT) by wounding (W) with a pattern wheel and application of oral secretions (OS) of Manduca sexta, but is impaired in the transgenic plants silenced in the expression of lipoxygenase 3 (LOX3) or MYB8 by RNAi with inverted-repeat (ir) constructs. The concentration of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) decreases in WT after W+OS, but the effects of jasmonic acid (JA) on N-investment into RuBisCO are unclear. Amino acids serve as precursors for putrescine and spermidine and for nicotinic acid (NA), which provide N for the synthesis of these metabolites. Amino acids are derived from nitrate (NO3−) reduction, followed by assimilation catalyzed by glutamine synthetase (GS) and glutamate synthase (GOGAT), and are also used as precursors for RuBisCO synthesis. JA-Ile=JA- isoleucine; NR=nitrate reductase; NiR=nitrite reductase. b) 15N-incorporation into roots, younger (yRL) and older rosette leaves (oRL) following pulse-labeling with K15NO3 27 days after germination was determined by isotope-ratio mass spectrometry (IRMS) (n=5). Grey arrows indicate the time-points of elicitation in the following experiments. During this time-frame 15N-incorporation was stable. At%=atomic percent.
Figure 2
Figure 2
Total N content in WT shoots decreases after simulated herbivory. N content of shoots of irLOX3, irMYB8 and WT (n=5) was determined by IRMS 4 days after the first W+OS elicitation. Unelicited plants were controls. Asterisks represent significant differences between treatments (*: p ≤ 0.05; n=5). Inset: The N content of WT roots (n = 5) was determined in a separate experiment at the same time-point. DM=dry mass
Figure 3
Figure 3
Silencing of LOX3 and MYB8 alters the N distribution between and within leaves. The N pools and total soluble protein (TSP) of leaves (oRL, yRL, S1=1st stem leaf) of irLOX3, irMYB8 and WT, calculated based on leaf mass. The N content was determined by IRMS and the TSP was measured by the Bradford assay. Plants were elicited as described for Figure 2. yRL and oRL were harvested 4 days after the first W+OS elicitation and when S1 leaves underwent the source-sink transition. Asterisks indicate differences among treatments (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001). Letters represent significant differences found using the minimum adequate model (n=5). For abbreviations see Figure 1.
Figure 4
Figure 4
Increased N-investment in nicotine, CP and DCS is accompanied by a decreased N-investment in protein. a) N-investment in residual TSP (TSP - (SSU + LSU)), RubisCO large (LSU) and small (SSU) subunit, nicotine, CP and DCS in oRL, yRL and S1 was calculated by multiplying the proportion of N in each compound with the concentration of the compound for each leaf. The amount of TSP was quantified by the Bradford assay, RuBisCO LSU and SSU were determined by LC-MSE and the defense metabolites by UPLC-UV-ToF-MS. Plants were elicited as described for Figure 2 and leaves were harvested as described for Figure 3 (n=5). FM=fresh mass. For other abbreviations see Figure 1. Heatmaps represent Kendall’s τ coefficient for pairwise correlation of N-investment in all of the above compounds among all genotype/elicitation groups. b) 15N-investment in RuBisCO LSU and SSU and defense metabolites was calculated as 15N-incorporation multiplied by the N-investment. Plants were pulse-labeled with K15NO3 3 days before the first treatment. 15N-incorporation was determined based on the MS-spectra with ProSipQuant (Taubert et al. 2011).
Figure 4
Figure 4
Increased N-investment in nicotine, CP and DCS is accompanied by a decreased N-investment in protein. a) N-investment in residual TSP (TSP - (SSU + LSU)), RubisCO large (LSU) and small (SSU) subunit, nicotine, CP and DCS in oRL, yRL and S1 was calculated by multiplying the proportion of N in each compound with the concentration of the compound for each leaf. The amount of TSP was quantified by the Bradford assay, RuBisCO LSU and SSU were determined by LC-MSE and the defense metabolites by UPLC-UV-ToF-MS. Plants were elicited as described for Figure 2 and leaves were harvested as described for Figure 3 (n=5). FM=fresh mass. For other abbreviations see Figure 1. Heatmaps represent Kendall’s τ coefficient for pairwise correlation of N-investment in all of the above compounds among all genotype/elicitation groups. b) 15N-investment in RuBisCO LSU and SSU and defense metabolites was calculated as 15N-incorporation multiplied by the N-investment. Plants were pulse-labeled with K15NO3 3 days before the first treatment. 15N-incorporation was determined based on the MS-spectra with ProSipQuant (Taubert et al. 2011).
Figure 5
Figure 5
Dynamics of 15N-incorporation into nicotine, CP, DCS and LSU demonstrates that recently assimilated N, not N derived from LSU metabolism, is rapidly invested into CP and DCS biosynthesis after elicitation. Three days before the first W+OS treatment plants were pulse labeled with K15NO3 (see Fig. 1a). The yRL at the time of labeling was harvested at indicated time points. 15N-incorporation (n=5) of RuBisCO LSU, nicotine, CP and DCS was determined as described for Figure 4. For abbreviations see Figure 1.
Figure 6
Figure 6
Herbivory-induced trade-offs of N-investment into growth and defense are mediated by MYB8. N-investment in defense causes a reallocation of N from the shoot to the root. We suggest that the transcription factor MYB8 probably via the synthesis of phenolamides (CP, DCS) is involved in the reallocation of N within the local leaf. N invested in phenolamides and in the root synthesized alkaloid nicotine increases after herbivory, while the N-investment in TSP and RuBisCO strongly decreases, but it is unlikely that N invested into phenolamides originates from RuBisCO metabolism. The height of the left and right side of the quadrangles represent relative changes in N-pool sizes for each compound of C and OS-elicited plants, respectively, using the N-amount of RuBisCO after elicitation as a reference (x). All N-pools within the depicted leaf show the ratios of measured values per mg fresh mass. Shoot, root and whole-leaf N-pools depicted outside the plant represent ratios of N determined per mg dry mass. For abbreviations see Figure 1.
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References

    1. Allen DK, Laclair RW, Ohlrogge JB, Shachar-Hill Y. Isotope labelling of Rubisco subunits provides in vivo information on subcellular biosynthesis and exchange of amino acids between compartments. Plant Cell Environ. 2012;35:1232–1244. - PMC - PubMed
    1. Allmann S, Halitschke R, Schuurink RC, Baldwin IT. Oxylipin channelling in Nicotiana attenuata: lipoxygenase 2 supplies substrates for green leaf volatile production. Plant Cell Environ. 2010;33:2028–2040. - PubMed
    1. Baldwin IT. Inducible nicotine production in native Nicotiana as an example of adaptive phenotypic plasticity. J. Chem. Ecol. 1999;25:3–30.
    1. Baldwin IT, Gorham D, Schmelz EA, Lewandowski CA, Lynds GY. Allocation of nitrogen to an inducible defense and seed production in Nicotiana attenuata. Oecologia. 1998;115:541–552. - PubMed
    1. Baldwin IT, Hamilton W. Jasmonate-induced responses of Nicotiana sylvestris results in fitness costs due to impaired competitive ability for nitrogen. J. Chem. Ecol. 2000;26:915–952.

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