- Email: [email protected]
Chemical Intervention: New Tools to Dissect Metabolic Signaling
TRPLSC 1533 No. of Pages 3
Spotlight
Chemical Intervention: New Tools to Dissect Metabolic Signaling Matthew Ramon1 and Filip Rolland2,* The metabolic intermediate trehalose-6-P (T6P) has emerged as a key regulator of plant growth and development, but the underlying mechanisms remain largely elusive. A recent publication reported a new chemical intervention strategy, providing a powerful tool to dissect T6P-mediated metabolic signaling.
Metabolic Signaling Gain- and loss-of-function mutants and transgenics are currently the most commonly used tools in basic plant research. However, while these approaches have been used successfully, their use can also be either complicated by ectopic and longterm feedback effects or limited in cases of where the loss of function leads to lethality and redundancy. These limitations are even more pronounced when studying pathways central to plant life. Particularly challenging has been the analysis of metabolic signaling because the functions of regulatory metabolites are intricately linked to their vital role as substrates in primary metabolism. Sugars, for example, were identified as signaling molecules for plant carbon and energy status, integrating both developmental and environmental cues [1,2]. Although mutant screens with exogenous application of relatively high levels of sugars have been useful for initial analyses, further elucidation of the mechanisms involved is hampered by nonspecific and indirect effects and their fast interconversion and metabolism. In addition, metabolic intermediates, such as sugar-
phosphates, are not readily taken up. Interestingly, a recent study [3] reported a new controlled chemical intervention strategy using plant-permeable precursors that release the active T6P signaling molecule in planta only after light activation, opening new possibilities for more controlled and targeted investigation.
Trehalose-6-P and the T6P– Sucrose Nexus T6P is an intriguing [83_TD$IF]but reluctant metabolite, only present in minute amounts in flowering plants. It is synthesized from glucose-6-P and UDP-glucose by T6P synthase (TPS) activity and converted to trehalose by T6P phosphatase (TPP) activity. The discovery of extended TPS and TPP gene families in all higher plant genomes [4,5] and the embryo lethality of TPS deficiency in [69_TD$IF]arabidopsis [6] revealed an important role for trehalose metabolism in higher plants. The remarkable opposite effects of transgenic modification of T6P levels by overexpression of bacterial TPS and TPP on plant growth subsequently confirmed a more specific role for the T6P intermediate [7]. However, the phenotypes are complex and the exact mechanisms of T6P action are still unclear. Another important complicating factor are the low (femtomole scale) levels of this sugar, [84_TD$IF]but optimized and validated approaches, such as anionexchange LC-MS/MS quantification, have revealed a strong positive correlation between T6P and sucrose levels. This indicated that T6P acts as a proxy for plant sucrose status [8]. Interestingly, sucrose regulation of T6P levels is not only mediated by passive mass-action effects at the substrate level, but also involves de novo protein synthesis [9]. Moreover, the persistent correlation over a range of growth conditions in transgenic plants with genetically altered T6P levels supports a sucrose–T6P nexus model: T6P not only signals sucrose availability, but in turn also negatively regulates sucrose levels by restricting sucrose synthesis and/or promoting sucrose consumption [9]. Consistently, T6P has
been implicated in the regulation of transitory starch turnover and other metabolic and carbon- and energy-requiring growth-related processes (reviewed in [10]) (Figure 1A). This makes it difficult to discriminate between the direct and indirect effects of T6P. The SNF1-related kinase 1 (SnRK1) protein kinase, the ortholog of yeast SNF1 and mammalian AMP-activated kinase and a central component of the plant metabolic signaling network, was identified as a target in developing tissues, where SnRK1 inhibition by T6P is dependent on an as-yetunidentified protein factor [11].
A Chemical Intervention Strategy The complex tissue-specific interactions and metabolic feedback mechanisms emphasize the need for more considerate approaches and new tools to dissect T6P-dependent and -independent sucrose effects and SnRK1-dependent and -independent T6P signaling. Griffiths et al. designed and synthesized T6P signaling precursors using different elegant phosphorus chemistries to mask charge and increase hydrophobicity (increasing permeability and tissue uptake), while enabling photo-activated controlled release of T6P in planta [3] (Figure 1B). Whereas transgenic intervention typically alters levels only [85_TD$IF]twofold to threefold, this strategy can increase endogenous plant T6P levels by up to 100-fold with high fluxes of ultraviolet (UV) light. Remarkably, treatment in [69_TD$IF]arabidopsis not only enhanced trehalose levels (indicative of an active metabolism of the released compounds) and reduced SnRK1 signaling (based on target gene expression), but also significantly increased sucrose synthesis. This is surprising because one would expect negative feedback regulation, and is possibly a consequence of nonphysiological high levels of T6P. This also resulted in an amplification effect because the increase in sucrose levels was associated with an (expected) rise in endogenous T6P production and starch biosynthesis [3].
Trends in Plant Science, Month Year, Vol. xx, No. yy
1
TRPLSC 1533 No. of Pages 3
(A)
(B) O
T6P-independent signaling
Hexose-P
Sucrose
P O O
O HO
Consumpon
PM
SnRK1 O
Hexose-P
TPS
O
P
TPP
Trehalose
O HO HO
T6P signaling
O
Extracellular
HO
T6P
O
P O
Compound 3
OH
O
OHO
O
O2N O2N
OH
O
HO HO
Intracellular
Uptake O
O
P O
HO
O
OH
O OH O
HO HO
OH
O
O
T6P
HO
OH
O OH O
HO
O
OH
HO
Photoacvaon
Figure 1. T[76_TD$IF]6P-Mediated Sugar Signaling. (A) The trehalose-6-phosphate (T6P[7_TD$IF])–sucrose nexus. Sucrose regulates T6P levels by stimulating T6P synthase (TPS)mediated biosynthesis and/or inhibition of further metabolisation to trehalose by the T6P phosphatases (TPP). T6P in turn regulates sucrose levels by restricting sucrose synthesis and/or promoting sucrose consumption [9]. T6P-dependent signaling of sucrose status is mediated by inhibition of SNF1-related kinase 1 (SnRK1) in growing tissues [11]. (B) Controlled chemical intervention with T6P precursor molecules. Plant-permeable precursor molecules are taken up and photoactivated with in planta release of T6P. The side chain of the most effective precursor (compound 3, produced using phosphoramidite chemistry) is shown [3].
Application in Crop Species Previous work had already shown that the spatiotemporally targeted reduction of T6P levels, using a specific promoter to drive TPP expression, can increase local sugar supply in developing maize ears [12], in line with the T6P–sucrose nexus model. Remarkably, in the recent study, treatment with the T6P analogs during the grain-filling period of a spring wheat variety in a controlled environment similarly increased grain size and starch content, most likely also due to an increase in sucrose levels [3]. This work demonstrates that the controlled chemical intervention strategy is also applicable to nonmodel crops, although the exact mode of action of the increased T6P levels is still puzzling, and application in more [86_TD$IF]variable field conditions might prove more challenging with densely growing plants and the associated reduced accessibility and shading of target tissues.
modification technologies used for basic research, which can also be applied in non-model crops that typically have more complex genomes and low transformation efficiencies. The possibility of temporal and spatial control (using the application of short pulses in specific conditions, tissues, and developmental stages) can also significantly reduce unwanted ectopic effects. In combination with cellular assays and other novel research tools, such as plant-optimized genetically encoded sensors for monitoring in vivo T6P dynamics and SnRK1 activity, this approach will enable the more direct investigation and elucidation of the molecular mechanisms and targets of T6P signaling. However, it is vital that physiologically relevant levels of active compounds (and activating light conditions) are used to avoid confounding effects.
So, while T6P remains an elusive molecule, controlled chemical intervention, A Powerful New Research Tool However, it is clear that controlled chemi- when used appropriately, provides a cal intervention is an important new addi- powerful new tool to investigate its function to the more elaborate genetic tions and the mechanisms involved. This
2
Trends in Plant Science, Month Year, Vol. xx, No. yy
recent study illustrates the potential of this strategy and also confirms T6P signaling as a key regulatory mechanism in crop species. Analogous strategies, targeting specific physiological processes, will undoubtedly accelerate future biological research and discoveries. Disclaimer M.R. is employed by the European Food Safety Authority (EFSA) in its GMO Unit, which provides scientific and administrative support to the GMO Panel. However, the present article is published under the sole responsibility of the author and may not be considered as an EFSA scientific output. The positions and opinions presented in this article are those of the author alone and are not intended to represent the views or scientific works of EFSA. To know about the views or scientific outputs of EFSA, please consult its website under www.efsa.europa.eu.
Acknowledgments Work in the Rolland lab is supported by grants from the Research Foundation - Flanders (FWO) and KU Leuven. We sincerely apologize to colleagues for not citing all relevant work due to space limitations. 1 2
European Food Safety Authority, [79_TD$IF]43126 Parma PR, Italy
Laboratory for Molecular Plant Biology, Biology Department, University of Leuven KU Leuven, [81_TD$IF]Leuven, Belgium
TRPLSC 1533 No. of Pages 3
*Correspondence: fi[email protected] (F. Rolland). http://dx.doi.org/10.1016/j.tplants.2017.03.006 References 1. Lastdrager, J. et al. (2014) Sugar signals and the control of plant growth and development. J. Exp. Bot. 65, 799–807 2. Sheen, J. (2014) Master regulators in plant glucose signaling networks. J. Plant Biol. 57, 67–79 3. Griffiths, C.A. et al. (2016) Chemical intervention in plant sugar signalling increases yield and resilience. Nature 540, 574–578 4. Avonce, N. et al. (2006) Insights on the evolution of trehalose biosynthesis. BMC Evol. Biol. 6, 109
5. Lunn, J.E. (2007) Gene families and evolution of trehalose metabolism in plants. Funct. Plant Biol. 34, 550–563 6. Eastmond, P.J. et al. (2002) Trehalose-6-phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation. Plant J. 29, 225–235 7. Schluepmann, H. et al. (2003) Trehalose 6-phosphate is indispensable for carbohydrate utilization and growth in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U.S.A. 100, 6849–6854 8. Lunn, J.E. et al. (2006) Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana. Biochem. J. 397, 139–148
9. Yadav, U.P. et al. (2014) The sucrose-trehalose 6-phosphate (Tre6P) nexus: specificity and mechanisms of sucrose signalling by Tre6P. J. Exp. Bot. 65, 1051–1068 10. Figueroa, C.M. and Lunn, J.E. (2016) A tale of two sugars: trehalose 6-phosphate and sucrose. Plant Physiol. 172, 7–27 11. Zhang, Y. et al. (2009) Inhibition of SNF1-related protein kinase1 activity and regulation of metabolic pathways by trehalose-6-phosphate. Plant Physiol. 149, 1860–1871 12. Nuccio, M.L. et al. (2015) Expression of trehalose-6-phosphate phosphatase in maize ears improves yield in wellwatered and drought conditions. Nat. Biotechnol. 33, 862–869
Trends in Plant Science, Month Year, Vol. xx, No. yy
3
Spotlight
Chemical Intervention: New Tools to Dissect Metabolic Signaling Matthew Ramon1 and Filip Rolland2,* The metabolic intermediate trehalose-6-P (T6P) has emerged as a key regulator of plant growth and development, but the underlying mechanisms remain largely elusive. A recent publication reported a new chemical intervention strategy, providing a powerful tool to dissect T6P-mediated metabolic signaling.
Metabolic Signaling Gain- and loss-of-function mutants and transgenics are currently the most commonly used tools in basic plant research. However, while these approaches have been used successfully, their use can also be either complicated by ectopic and longterm feedback effects or limited in cases of where the loss of function leads to lethality and redundancy. These limitations are even more pronounced when studying pathways central to plant life. Particularly challenging has been the analysis of metabolic signaling because the functions of regulatory metabolites are intricately linked to their vital role as substrates in primary metabolism. Sugars, for example, were identified as signaling molecules for plant carbon and energy status, integrating both developmental and environmental cues [1,2]. Although mutant screens with exogenous application of relatively high levels of sugars have been useful for initial analyses, further elucidation of the mechanisms involved is hampered by nonspecific and indirect effects and their fast interconversion and metabolism. In addition, metabolic intermediates, such as sugar-
phosphates, are not readily taken up. Interestingly, a recent study [3] reported a new controlled chemical intervention strategy using plant-permeable precursors that release the active T6P signaling molecule in planta only after light activation, opening new possibilities for more controlled and targeted investigation.
Trehalose-6-P and the T6P– Sucrose Nexus T6P is an intriguing [83_TD$IF]but reluctant metabolite, only present in minute amounts in flowering plants. It is synthesized from glucose-6-P and UDP-glucose by T6P synthase (TPS) activity and converted to trehalose by T6P phosphatase (TPP) activity. The discovery of extended TPS and TPP gene families in all higher plant genomes [4,5] and the embryo lethality of TPS deficiency in [69_TD$IF]arabidopsis [6] revealed an important role for trehalose metabolism in higher plants. The remarkable opposite effects of transgenic modification of T6P levels by overexpression of bacterial TPS and TPP on plant growth subsequently confirmed a more specific role for the T6P intermediate [7]. However, the phenotypes are complex and the exact mechanisms of T6P action are still unclear. Another important complicating factor are the low (femtomole scale) levels of this sugar, [84_TD$IF]but optimized and validated approaches, such as anionexchange LC-MS/MS quantification, have revealed a strong positive correlation between T6P and sucrose levels. This indicated that T6P acts as a proxy for plant sucrose status [8]. Interestingly, sucrose regulation of T6P levels is not only mediated by passive mass-action effects at the substrate level, but also involves de novo protein synthesis [9]. Moreover, the persistent correlation over a range of growth conditions in transgenic plants with genetically altered T6P levels supports a sucrose–T6P nexus model: T6P not only signals sucrose availability, but in turn also negatively regulates sucrose levels by restricting sucrose synthesis and/or promoting sucrose consumption [9]. Consistently, T6P has
been implicated in the regulation of transitory starch turnover and other metabolic and carbon- and energy-requiring growth-related processes (reviewed in [10]) (Figure 1A). This makes it difficult to discriminate between the direct and indirect effects of T6P. The SNF1-related kinase 1 (SnRK1) protein kinase, the ortholog of yeast SNF1 and mammalian AMP-activated kinase and a central component of the plant metabolic signaling network, was identified as a target in developing tissues, where SnRK1 inhibition by T6P is dependent on an as-yetunidentified protein factor [11].
A Chemical Intervention Strategy The complex tissue-specific interactions and metabolic feedback mechanisms emphasize the need for more considerate approaches and new tools to dissect T6P-dependent and -independent sucrose effects and SnRK1-dependent and -independent T6P signaling. Griffiths et al. designed and synthesized T6P signaling precursors using different elegant phosphorus chemistries to mask charge and increase hydrophobicity (increasing permeability and tissue uptake), while enabling photo-activated controlled release of T6P in planta [3] (Figure 1B). Whereas transgenic intervention typically alters levels only [85_TD$IF]twofold to threefold, this strategy can increase endogenous plant T6P levels by up to 100-fold with high fluxes of ultraviolet (UV) light. Remarkably, treatment in [69_TD$IF]arabidopsis not only enhanced trehalose levels (indicative of an active metabolism of the released compounds) and reduced SnRK1 signaling (based on target gene expression), but also significantly increased sucrose synthesis. This is surprising because one would expect negative feedback regulation, and is possibly a consequence of nonphysiological high levels of T6P. This also resulted in an amplification effect because the increase in sucrose levels was associated with an (expected) rise in endogenous T6P production and starch biosynthesis [3].
Trends in Plant Science, Month Year, Vol. xx, No. yy
1
TRPLSC 1533 No. of Pages 3
(A)
(B) O
T6P-independent signaling
Hexose-P
Sucrose
P O O
O HO
Consumpon
PM
SnRK1 O
Hexose-P
TPS
O
P
TPP
Trehalose
O HO HO
T6P signaling
O
Extracellular
HO
T6P
O
P O
Compound 3
OH
O
OHO
O
O2N O2N
OH
O
HO HO
Intracellular
Uptake O
O
P O
HO
O
OH
O OH O
HO HO
OH
O
O
T6P
HO
OH
O OH O
HO
O
OH
HO
Photoacvaon
Figure 1. T[76_TD$IF]6P-Mediated Sugar Signaling. (A) The trehalose-6-phosphate (T6P[7_TD$IF])–sucrose nexus. Sucrose regulates T6P levels by stimulating T6P synthase (TPS)mediated biosynthesis and/or inhibition of further metabolisation to trehalose by the T6P phosphatases (TPP). T6P in turn regulates sucrose levels by restricting sucrose synthesis and/or promoting sucrose consumption [9]. T6P-dependent signaling of sucrose status is mediated by inhibition of SNF1-related kinase 1 (SnRK1) in growing tissues [11]. (B) Controlled chemical intervention with T6P precursor molecules. Plant-permeable precursor molecules are taken up and photoactivated with in planta release of T6P. The side chain of the most effective precursor (compound 3, produced using phosphoramidite chemistry) is shown [3].
Application in Crop Species Previous work had already shown that the spatiotemporally targeted reduction of T6P levels, using a specific promoter to drive TPP expression, can increase local sugar supply in developing maize ears [12], in line with the T6P–sucrose nexus model. Remarkably, in the recent study, treatment with the T6P analogs during the grain-filling period of a spring wheat variety in a controlled environment similarly increased grain size and starch content, most likely also due to an increase in sucrose levels [3]. This work demonstrates that the controlled chemical intervention strategy is also applicable to nonmodel crops, although the exact mode of action of the increased T6P levels is still puzzling, and application in more [86_TD$IF]variable field conditions might prove more challenging with densely growing plants and the associated reduced accessibility and shading of target tissues.
modification technologies used for basic research, which can also be applied in non-model crops that typically have more complex genomes and low transformation efficiencies. The possibility of temporal and spatial control (using the application of short pulses in specific conditions, tissues, and developmental stages) can also significantly reduce unwanted ectopic effects. In combination with cellular assays and other novel research tools, such as plant-optimized genetically encoded sensors for monitoring in vivo T6P dynamics and SnRK1 activity, this approach will enable the more direct investigation and elucidation of the molecular mechanisms and targets of T6P signaling. However, it is vital that physiologically relevant levels of active compounds (and activating light conditions) are used to avoid confounding effects.
So, while T6P remains an elusive molecule, controlled chemical intervention, A Powerful New Research Tool However, it is clear that controlled chemi- when used appropriately, provides a cal intervention is an important new addi- powerful new tool to investigate its function to the more elaborate genetic tions and the mechanisms involved. This
2
Trends in Plant Science, Month Year, Vol. xx, No. yy
recent study illustrates the potential of this strategy and also confirms T6P signaling as a key regulatory mechanism in crop species. Analogous strategies, targeting specific physiological processes, will undoubtedly accelerate future biological research and discoveries. Disclaimer M.R. is employed by the European Food Safety Authority (EFSA) in its GMO Unit, which provides scientific and administrative support to the GMO Panel. However, the present article is published under the sole responsibility of the author and may not be considered as an EFSA scientific output. The positions and opinions presented in this article are those of the author alone and are not intended to represent the views or scientific works of EFSA. To know about the views or scientific outputs of EFSA, please consult its website under www.efsa.europa.eu.
Acknowledgments Work in the Rolland lab is supported by grants from the Research Foundation - Flanders (FWO) and KU Leuven. We sincerely apologize to colleagues for not citing all relevant work due to space limitations. 1 2
European Food Safety Authority, [79_TD$IF]43126 Parma PR, Italy
Laboratory for Molecular Plant Biology, Biology Department, University of Leuven KU Leuven, [81_TD$IF]Leuven, Belgium
TRPLSC 1533 No. of Pages 3
*Correspondence: fi[email protected] (F. Rolland). http://dx.doi.org/10.1016/j.tplants.2017.03.006 References 1. Lastdrager, J. et al. (2014) Sugar signals and the control of plant growth and development. J. Exp. Bot. 65, 799–807 2. Sheen, J. (2014) Master regulators in plant glucose signaling networks. J. Plant Biol. 57, 67–79 3. Griffiths, C.A. et al. (2016) Chemical intervention in plant sugar signalling increases yield and resilience. Nature 540, 574–578 4. Avonce, N. et al. (2006) Insights on the evolution of trehalose biosynthesis. BMC Evol. Biol. 6, 109
5. Lunn, J.E. (2007) Gene families and evolution of trehalose metabolism in plants. Funct. Plant Biol. 34, 550–563 6. Eastmond, P.J. et al. (2002) Trehalose-6-phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation. Plant J. 29, 225–235 7. Schluepmann, H. et al. (2003) Trehalose 6-phosphate is indispensable for carbohydrate utilization and growth in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U.S.A. 100, 6849–6854 8. Lunn, J.E. et al. (2006) Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana. Biochem. J. 397, 139–148
9. Yadav, U.P. et al. (2014) The sucrose-trehalose 6-phosphate (Tre6P) nexus: specificity and mechanisms of sucrose signalling by Tre6P. J. Exp. Bot. 65, 1051–1068 10. Figueroa, C.M. and Lunn, J.E. (2016) A tale of two sugars: trehalose 6-phosphate and sucrose. Plant Physiol. 172, 7–27 11. Zhang, Y. et al. (2009) Inhibition of SNF1-related protein kinase1 activity and regulation of metabolic pathways by trehalose-6-phosphate. Plant Physiol. 149, 1860–1871 12. Nuccio, M.L. et al. (2015) Expression of trehalose-6-phosphate phosphatase in maize ears improves yield in wellwatered and drought conditions. Nat. Biotechnol. 33, 862–869
Trends in Plant Science, Month Year, Vol. xx, No. yy
3