Chemical intervention in plant sugar signalling increases yield and resilience

Article abstract of DOI:10.1038/nature20591

The pressing global issue of food insecurity due to population growth, diminishing land and variable climate can only be addressed in agriculture by improving both maximum crop yield potential and resilience. Genetic modification is one potential solution, but has yet to achieve worldwide acceptance, particularly for crops such as wheat. Trehalose-6-phosphate (T6P), a central sugar signal in plants, regulates sucrose use and allocation, underpinning crop growth and development. Here we show that application of a chemical intervention strategy directly modulates T6P levels in planta. Plant-permeable analogues of T6P were designed and constructed based on a 'signalling-precursor' concept for permeability, ready uptake and sunlight-triggered release of T6P in planta. We show that chemical intervention in a potent sugar signal increases grain yield, whereas application to vegetative tissue improves recovery and resurrection from drought. This technology offers a means to combine increases in yield with crop stress resilience. Given the generality of the T6P pathway in plants and other small-molecule signals in biology, these studies suggest that suitable synthetic exogenous small-molecule signal precursors can be used to directly enhance plant performance and perhaps other organism function.

Full text of DOI:10.1038/nature20591

LETTER
doi:10.1038/nature20591
Chemical intervention in plant sugar signalling
increases yield and resilience
Cara A. Griffiths1*, Ram Sagar2*†, Yiqun Geng2*, Lucia F. Primavesi1, Mitul K. Patel2, Melissa K. Passarelli3, Ian S. Gilmore3,
Rory T. Steven3, Josephine Bunch3,4, Matthew J. Paul1 & Benjamin G. Davis2
The pressing global issue of food insecurity due to population dependent on both light intensity and frequency under a range of
growth, diminishing land and variable climate can only be addressed conditions. Consistent with design, light-sensitive groups were differently
in agriculture by improving both maximum crop yield potential susceptible. Precursor ortho-nitrobenzyl (oNB)-T6P 1, for
and resilience1,2. Genetic modification is one potential solution, example, generated T6P by light-activated release more rapidly
but has yet to achieve worldwide acceptance, particularly for crops at lower wavelengths, while compound mono-dimethoxy(ortho-
such as wheat3. Trehalose-6-phosphate (T6P), a central sugar signal nitro)benzyl (mono-DMNB)-T6P 4 was more reactive at higher
in plants, regulates sucrose use and allocation, underpinning wavelengths. Although release with higher light intensity/photon flux
crop growth and development4,5. Here we show that application (125 W/365μmol m−2 s−1 (with photon flux defined as the number
of a chemical intervention strategy directly modulates T6P levels of photons per m2 per second) compared to 8 W/23μmol m−2 s−1
)
in planta. Plant-permeable analogues of T6P were designed was more rapid, direct sunlight proved sufficient, in some cases
and constructed based on a ‘signalling-precursor’ concept for resulting in t95 as brief as 90min (for (ortho-nitrophenyl)ethyl (oNPE)-
permeability, ready uptake and sunlight-triggered release of T6P T6P (compound 3)). Nuclear magnetic resonance spectroscopy analysis
in planta. We show that chemical intervention in a potent sugar (Supplementary Methods and Extended Data Fig. 1e, f) confirmed that
signal increases grain yield, whereas application to vegetative tissue T6P was formed and that potent inhibitory activity against SnRK1 was
improves recovery and resurrection from drought. This technology induced (Extended Data Fig. 2).
offers a means to combine increases in yield with crop stress
Following successful in vitro release, uptake in planta was examined.
resilience. Given the generality of the T6P pathway in plants and Compounds 1–4 (at a final concentration of 1mM) were fed to roots of
other small-molecule signals in biology, these studies suggest that plantlets of Arabidopsis thaliana and the aerial parts were analysed over
suitable synthetic exogenous small-molecule signal precursors can time and with increasing dose (Fig. 2 and Supplementary Tables 2–7).
be used to directly enhance plant performance and perhaps other High-performance liquid chromatography (HPLC) followed by quanti-
organism function.
tative mass spectrometry14 (HPLC–MS) of extracts of the aboveground
We designed a signalling-precursor strategy on the basis of release by biomass (shoot and leaves) showed increasing uptake of the compounds
light (Extended Data Fig. 1). Light-activated control is a potent modu- over time (Fig. 2a and Extended Data Fig. 3) and with increasing dose.
lation strategy in biology, allowing for temporal and spatial resolution Consistent with design, structural variation showed that altered hydro-
surpassing that of standard genetic methods6. Additionally, such phobicity modulated permeability15 and transport16. Notably, systematic
resolution can be increased when combined with small-molecule variation of group type and copy number identified compound 3
chemical control7–9. Potency is further increased when releasing a sig- (log[P] of the compound is 0.11 0.60, where P is the partition
nalling molecule, as the effects of light-activated control are increased coefficient; see also Supplementary Methods and Supplementary Table 17)
several-fold through the inherent amplification of signalling.
as the most readily taken up (Fig. 2a), with absorption of approxi-
Hydrophilic or charged molecules do not readily enter plants unless mately 20% after 72h. Compounds 1, 2 (dimethoxy(ortho-nitro)benzyl
transported. We therefore designed unnatural precursors (1–4) of (DMNB)-T6P) and 4 (log[P] of the compounds ranged from −2.35
T6P with groups to mask charge, increase hydrophobicity and also to −0.17), on the other hand, were less readily taken up.
to induce release by light (Fig. 1a). Their construction (Fig. 1b) used
Next, we investigated the light-activated release in planta. Plants were
different phosphorus chemistries: phosphoramidite chemistry10,11 to treated with compounds dissolved in medium, grown for a further
create P(III)-intermediates that were then oxidized to correspond- three days, irradiated, and the shoots were harvested and extracted17.
ing P(V)-phosphotriesters, or direct P(V)-phosphorylation chem- T6P release was confirmed by tandem mass spectrometry (Extended
istry (Fig. 1b). Regioselective access to the OH-6 group in trehalose Data Fig. 4) and determined by quantitative HPLC–MS (with 2-deoxy-
exploited trimethylsilyl as a protecting group that is chemically orthog- glucose-6-phosphate as an internal standard14; Fig. 2b, Extended
onal to the phosphotriester; 12 was prepared on a multigram scale12. Data Fig. 4 and Supplementary Table 10). Release in planta could be
Phosphorylation (reaction with phosphoramidites 9–11 (refs 10, 11) controlled and modulated by the choice of light source and signalling
followed by tBuOOH, or treatment with POCl3 (ref. 13) followed precursor (Fig. 2b and Supplementary Table 10).
by the addition of the appropriate alcohol) gave intermediates that
Most transgenic approaches only alter T6P over a 2–3-fold range4,5
.
were deprotected under mildly acidic conditions (see Supplementary Using compounds 1–4, levels of up to 900nmol g−1 fresh weight were
Methods). 1–4 were all inactive against SnRK1 (SNF1-related kinase 1) attainable, which was 100-fold higher than endogenous levels and
(Extended Data Fig. 2).
75-fold higher than can be achieved with genetic methods. Consistent
Mass spectrometry, thin-layer chromatography and nuclear magnetic with this strategy, maximal T6P was released when precursor-treated
resonance spectroscopy (see Supplementary Methods, Supplementary plants were irradiated with the highest flux (100 W/292μmol m−2 s−1
Table 1 and Extended Data Fig. 1) showed release times (95% release, t95) UV) in all cases. Notably, and with relevance to application in the field,
1Plant Biology and Crop Science, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK. 2Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road,
Oxford OX1 3TA, UK. 3National Centre of Excellence in Mass Spectrometry Imaging (NiCE-MSI), National Physical Laboratory (NPL), Teddington, Middlesex TW11 0LW, UK. 4School of Pharmacy,
University of Nottingham, Nottingham NG7 2RD, UK. †Present address: Department of Chemistry, School of Natural Sciences, Shiv Nadar University, Greater Noida 201 314, India.
*These authors contributed equally to this work.
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RESEARCH LETTER
MeO
OMe
a
b
O
O
O
O
O
O
P
O
P
O
–O
O
O
O
O
O
O
O
=
O2N
O2N
O2N
O2N
O
O
O2N
O2N
O
O
HO
P
O
P
O
P
O
O
P
O
O2N
OH
OH
O
O
OH
HO
HO
HO
1–4
MeO
OMe
MeO
OMe
1
2
3
4
R″
R″
R″
R″
R″
R″
Et3N/THF, –20°C to RT
2–19 h,70–88%
O
Cl
O
O
O
O
(i) Tetrazole,
CH2Cl2 or CH3CN,
0–5°C, 2–4 h
O2N
O2N R′
NO2
NO2
R′
R′
R′
N
P
N
P
P
O
+
Cl
O
HO
OH
OH
HO
NO2
O
O
(ii) tBuOOH, 30min
OH
1–3
R′
R′ = H or CH3, R″ = H or OCH3
HO
OH
R″
R″
R″
R″
(iii) Dowex-H+, MeOH, RT,
1 h, 56–87% over 3 steps
5
9–11
HO
6–8
MeO
OMe
O
O
O2N
(i) POCl3/pyridine, 10 min
then DMNB-OH, 1 h
P
O
[Si]O
[Si]O
HO
[Si]O
O
[Si]O
O
HO
O
K2CO3/MeOH
0°C, 1 h
HO
O
O[Si]
O[Si]
O
O[Si]
O[Si]
O
OH
OH
O
O
O[Si]
O
O[Si]
(ii) Dowex-H+, MeOH, 1 h
62% over 2 steps
OH
[Si]O
O[Si]
O[Si]
HO
OH
[Si]O
[Si] = (CH3 3Si
[Si]O
4
HO
)
12
Figure 1 | Design and synthesis of signalling precursors of T6P. T6P
is plant-impermeable; synthesis of plant-permeable variants allowed
subsequent photo-activated release of T6P in planta. a, Designed
precursors 1–4, with the backbone on the left and side chains (blue circle
on the back bone) of the individual precursors on the right. b, Synthesis
of the precursors using phosphoramidite chemistry (1–3) or direct
phosphorylation chemistry (4) from the key intermediate compound 12.
Universally 13C-labelled 2* was prepared in essentially the same manner
(see Extended Data Fig. 7a). THF, tetrahydrofuran.
with sunlight only, all treated plants released significantly increased also through the use of unnaturally enriched isotopic labelling of the
amounts of T6P (39–296nmol g−1 fresh weight) (Supplementary Table signalling precursors, allowing for unambiguous delineation of their
10), some approximately 4–30-fold above endogenous levels. There fate (Fig. 4, Extended Data Fig. 6 and Supplementary Methods). Thus,
was no significant reduction in the fresh weight of plantlets treated in 7-day-old A. thaliana seedlings21, treatment with 1mM of compound
with 1mM of compound (Supplementary Methods and Extended Data 2 or 3, fed for 24h before exposure to light (UV 8 W/23μmol m−2 s−1),
Fig. 5), suggesting low toxicity. Accumulation of T6P after treatment led to peak T6P after 60 min (229 and 159 nmol g−1 fresh weight,
was analysed with mass spectrometry imaging18 (Fig. 3) using signa- respectively), which declined over the following 2 days (Fig. 4a).
ture-ion markers19 in treated leaves of A. thaliana seedlings (Fig. 3b) Corresponding trehalose levels were also elevated, with peaks at around
after 2h of irradiation; the different distributions from compounds 2 2h (up to a maximum of 134nmol g−1 fresh weight compared to control
and 3 appeared consistent with their measured release rates. Notably, levels of 20nmol g−1 fresh weight; Fig. 4b), confirming the metabolism
increased trehalose was also observed20 in the same regions (Fig. 3c), indicated by mass spectrometry imaging. Glucose, the next sugar in
suggesting metabolism. Moreover, mass spectrometry imaging using the pathway, was also increased but to a smaller degree, peaking at
treatment-specific ions corroborated the uptake of precursors into around 2–4h (Fig. 4d). These levels are consistent with known low
leaves (Fig. 3d, e).
metabolic fluxes22. Given known interrelationships5, sucrose levels
The dynamics of this enhanced in planta T6P release, and possible were also determined. Notably, these increased 2–3-fold over the first
consequent metabolic products, were determined not only through 2h of irradiation (Fig. 4c) and correlated positively with T6P for both
both quantitative HPLC–MS and/or enzymatic quantification, but compounds 2 and 3 (Fig. 4); whereas fructose was minimally affected
a
b
otsA transgenic
Water
1
2
3
4
1,000
800
12
10
8
1
2
3
4
600
300
200
100
0
6
**
4
2
*
**
**
**
*
*
0
otsA
GL
8 h
GL
GL +
Sunlight
8 h
GL +
UV 100 W
8 h
24 h
48 h
72 h
2 × 8 h UV 8 W
2 × 8 h
292 μmol m−2 s−1; sunlight (cloud), 250 μmol m−2 s−1; and sunlight
(no cloud), 1,440 μmol m−2 s−1. ANOVA showed significant differences
(P <0.001) between treatments (water or precursor) for each regime.
All treatments with precursor and UV showed significance (P <0.001,
least significant difference (LSD)) compared to water and UV. For growth
light irradiance *P <0.05; **P <0.01 (LSD, data shown on a linear scale).
See also Supplementary Table 10.
Figure 2 | In planta uptake of signalling precursors and T6P release.
a, Uptake of compounds 1–4 (1 mM in medium) at 24, 48 and 72 h
(data are shown as mean s.e.m., n =3). FW, fresh weight. b, T6P released
in planta (data are shown as mean s.e.m., n =3). Compounds were
applied 18 days after sowing and then irradiated for 72 h. otsA, A. thaliana
overexpressing trehalose-phosphate synthase. Growth light (GL) and other
conditions are defined as the following levels of photon flux: GL, 250 μmol
m
−2 s−1; GL +8 W UV (365 nm), 23 μmol m−2 s−1; GL +100 W UV,
2
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LETTER RESEARCH
a
b
1.0 × 10–4
Control
3-treated leaf
2-treated leaf
Control
5.0 × 10–5
0
30
20
80
150
160
170
170
170
170
180
174.8
190
200
198.8
210
220
1.0 × 10–4
5.0 × 10–5
0
196.8
158.8
3
2
212.8
214.7
150
160
180
174.8
190
200
210
220
1.0 × 10–4
5.0 × 10–5
0
196.8
156.8
212.8
214.8
0
0
m/z 18
0
158.9
160
198.8
T6P
markers
m/z 27.9
1 mm
c
3-treated leaf
1 mm
1 mm
150
180
174.9
180
190
200
210
210
220
1.0 × 10–3
5.0 × 10–4
0
156.9
212.8
198.8
T6P
reference
196.8
214.8
158.9
160
150
190
200
220
m/z
d
160
120
80
40
0
Control
3
2
30
20
100
T6P
Trehalose
0
Triacontanoic acid
(melissic acid)
markers
0.5 mm
0
0
e
63
200
400
15
600
800
10
1,000
0.6
m/z 237
m/z 467
0
411
0
0
0
236.8 237 236.2
467
467.5
505.5 506 506.5
m/z
0
m/z
m/z
Control
3-treated leaf 2-treated leaf
186
m/z 506
0
Figure 3 | Mass spectometry imaging of treated leaves. For all
green, positive mode), trehalose (m/z 325.2, 321.2, blue, negative mode)
and overlay with known19 epicuticular wax markers (red, negative mode)
in the 3-treated leaf. d, e, Matrix-assisted laser desorption/ionization–
mass spectometry (MALDI–MS). d, Overlay of mean on-leaf spectra for
control, 3-treated and 2-treated leaves. Lower panels show expansions for
correlated markers. e, RGB-colour overlay images of marker ions; separate
ion images are shown on right. Images in b, c and e are representative of
three individual images.
ion images, the pixel intensity scale represents the area under the
corresponding m/z peak. a, Time-of-flight secondary ion mass
spectrometry (ToF–SIMS) spectra from the surface of A. thaliana leaves
(top three spectra). Marker ions m/z 156.8, 196.8 and 212.8 in T6P
reference (bottom spectrum). b, T6P (three markers, green) in control
(left), 3-treated (middle) and 2-treated (right) leaves, anti-colocalized
with H2O (m/z 18.0) and silicon substrate (m/z 27.9). c, T6P (markers,
(Extended Data Fig. 6d). We confirmed that inhibited growth was not as sucrose induces T6P5,21. Together these data suggest that perturba-
an explanation for sucrose accumulation and found instead that growth tion of T6P levels occurs via two modes of action: direct release from
was stimulated by T6P (Extended Data Fig. 6e).
the signalling precursor and simultaneously induced biosynthesis of
Creation of a 13C-isotopically-labelled variant 2* (Extended Data T6P by the plant.
Fig. 7a) allowed direct tracking via ‘mass-shifts’ of the corresponding
Our data indicate that compound 3 is the plant-permeable sig-
ions using mass spectrometry. Treatment with 2* led to release of nalling precursor with the greatest tissue uptake coupled with the
13C-T6P and consequent sequential metabolism (to 13C-trehalose and greatest temporal control (consistent with its tuned permeability
13C-glucose) following essentially the same dynamics (Extended Data and its fastest release rates), allowing minimal application amounts
Fig. 7). Notably, using this method, mass labelling also showed that the (0.1mM), while still able to enhance T6P levels around 1.5–6.5-fold
compounds not only released, but also induced T6P. This accounted for above endogenous levels without potentially detrimental disrup-
approximately half of T6P measured at 30min, thereby providing direct tion of metabolism23. Plants were treated with precursor 3 for 72h
evidence of induction of de novo T6P synthesis and this induction con- and were then subjected to a single 8-h period under growth lights
tinued, giving rise to increased T6P accumulation over time (Extended supplemented with 8 W UV (Supplementary Table 10, generating
Data Fig. 7). This could be due to the large increase in sucrose observed, around 21 nmol g−1 fresh weight T6P) and harvested a day later.
Figure 4 | In planta T6P release and sugar
metabolism over time. a–d, Seven-day-old A.
thaliana seedlings grown in liquid culture were
treated with 1 mM of either 3 or 2; control seedlings
were treated with water. Seedlings were left under
growth lights to take up the signalling precursors
for 24 h, after which the plants were exposed to
23 μmol.m2 s−1 UV for 2 h. Measurements were
taken 1 d after uptake (pre-UV), 30, 60 and
120 min after the initiation of UV treatment
(23 μmol m−2 s−1), and 1 and 2 days after the
initiation of UV treatment. a, T6P content.
b, Trehalose content. c, Sucrose content. d, Glucose
content. In all cases n =3; *P <0.05; **P <0.01;
Student’s t-test; data are shown as mean s.e.m.
a
b
Control
3
2
300
250
200
150
100
50
180
160
140
120
100
80
*
**
**
*
*
**
*
*
**
*
**
*
*
*
60
*
*
40 **
20
*
0
0
Pre-UV 30 min 60 min 120 min
1 d
2 d
Pre-UV 30 min 60 min 120 min
1 d
2 d
c
d
40
35
30
25
20
15
10
5
10
**
*
**
8
6
4
2
0
**
*
**
*
*
**
*
*
**
*
*
**
*
0
Pre-UV 30 min 60 min 120 min
1 d
2 d
Pre-UV 30 min 60 min 120 min
1 d
2 d
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RESEARCH LETTER
a
The mean starch level (63.2μmol g−1 fresh weight) determined24 was
significantly higher (F(1,14) =13.59; P=0.002) than for water-treated
plants (40.7μmol g−1 fresh weight; Extended Data Fig. 5d).
Controls
Signalling precursors
The production of T6P from compounds 1–4 involves fragmentation
with concomitant release of side products. Although considered25 to
be non-toxic, we nevertheless tested for any unexpected phenotypic
changes. Glucose-6-phosphate (G6P) analogues 14–17 of compounds
1–4 were synthesized (Supplementary Methods and Extended Data
Fig. 8) and compared for their activity; G6P-methyl-glycoside itself
is inactive in planta and in all interactions with SnRK1 (Extended
Data Fig. 8) and so its light-activated release from analogues 14–17
provided a useful control. Analogues 14–17 showed similar light-
activated release parameters to compounds 1–4 (Supplementary
Table 11) and relative uptake performance was similarly dependent
on the identity of the light-sensitive moiety (Extended Data Fig. 8 and
Supplementary Table 12–14). No toxicity was observed in any of the
plants treated with up to 0.5mM of analogues 14–17 (Supplementary
Methods and Extended Data Fig. 5a–c), suggesting that the light-
released moiety is benign. Critically, starch was not affected in controls
treated with compound 16, the G6P analogue of T6P precursor 3.
The rate of starch synthesis in A. thaliana over a 12-h period (Extended
Data Fig. 5f, g) indicated a flux (0.037μmol min−1 g−1 fresh weight)
nearly three times that of water-treated controls (0.013μmol min−1 g−1
fresh weight). T6P is proposed to stimulate starch synthesis through
redox activation26 of ADP-glucose pyrophosphorylase (AGPase),
b
c
Ears
Whole plant
800
700
600
500
400
300
200
100
0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
Ears
Whole plant
*
*
*
*
*
*
*
Water
+T6P
+ Tre
+ 3
+ 2
Water
+T6P
+ Tre
+ 3
+ 2
(1 mM) (1 mM) (1 mM) (1 mM)
( 1mM) (1 mM) (1 mM) (1 mM)
Figure 5 | Increased crop yield. a, Increased grain size (20 per tube) after
spraying. b, Grain yield per plant with 1 mM of compounds 2 or 3.
c, Starch content of the grain. *P <0.05 compared to water control
(Student’s t-test). Data are shown as mean s.e.m. (n =6).
These data from A. thaliana raise the noteworthy possibility of
a rate-limiting enzyme. While not necessarily causal, consistent with enhanced starch synthesis in crops, potentially providing increased
this hypothesis, plants treated with compound 3 had significantly yield. Signalling precursors 2 and 3 were applied (0.1, 1 and 10mM)
higher AGPase activity (increased by 35%, Extended Data Fig. 5e). to spring wheat (Triticum aestivum Cadenza), which was grown in a
AGPase has been previously shown to affect starch turnover27.
controlled environment representative of summer in northern Europe.
We also measured the number of transcripts of genes known to Spraying occurred either to ears only or to the whole plant during the
be associated with T6P. First, SnRK1 is a proposed target of T6P30. grain-filling period (5, 10, 15 and 20 days post-anthesis (the flowering
SnRK1-induced (TPS5, bZIP11 (also known as GBF6) and UDPGDH period, DPA)) at mid-photoperiod. This increased grain yield per plant
(At3g29360)) and -repressed (TPS8 and ASN1) markers responded due to the formation of larger grain, particularly in plants treated with
synchronously to the activation of the precursors in a manner con- 1mM of compounds 2 or 3 (Fig. 5a, b). In these grains, starch content
sistent with known effects of T6P on SnRK1 activity (Extended Data increased 13–20% (Fig. 5c). A trend towards higher levels of starch
Fig. 9). However, other markers (for example, UDPGDH (At3g29360) and protein, when expressed as a percentage of component content
and bGAL4) showed clear temporal delay (Extended Data Fig. 9b); the per gram of grain, was also observed (Supplementary Table 16).
observed synchronization of these ‘secondary markers’ only occurred Dose–response analysis showed that yield peaked at a precursor
after a day, which suggests that these could be later, downstream targets concentration of 1 mM (Extended Data Fig. 10f). Minimal spray
of T6P. Second, starch is also a proposed target of T6P26,31. As starch amounts at only 10 DPA increased yield substantially at 1 and 10mM
levels were increased as a result of treatment, expression of starch doses (Extended Data Fig. 10g). Plants treated with compounds 2 and 3
biosynthetic genes was also analysed. Transcripts of APL3, SS3, BE1 stayed greener for longer than plants treated with water only, consistent
(also known as EMB2729) and GBSS1 were increased up to fivefold with chlorophyll content (Extended Data Fig. 10a, b) and previous
(Extended Data Fig. 9c).
observations for genetically-enhanced T6P content28. T6P release
Figure 6 | Increased crop resilience. a, Plants
after 20 d recovery following one application of
1 mM 3 or 2 1 d before rewatering. b, Dry weight
(DWT) biomass from plants in a. c, Plants after
one application of 1 mM 3 or 2 one day before
rewatering, cut at 5 d after rewatering, and left to
regrow for 10 d. White arrow and line, cut back
point. d, Fresh weight (FW) biomass of regrowth
from c. c, d, *P <0.05 compared to water-treated
control (Student’s t-test, n =6). Data are shown
as mean s.e.m.
a
c
Control
1mM 3
1mM 2
Control
1mM 3
1mM 2
b
7
6
5
4
3
2
1
0
d
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
*
Control
1 mM 3
1 mM 2
*
*
*
Control
1mM 3
1mM 2
0
0
5
10
15
20
25
30
Growth time (days)
4
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LETTER RESEARCH
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(1992).
14. Yang, M., Brazier, M., Edwards, R. & Davis, B. G. High-throughput mass-
spectrometry monitoring for multisubstrate enzymes: determining the kinetic
parameters and catalytic activities of glycosyltransferases. ChemBioChem 6,
346–357 (2005).
in the wheat grains treated with compounds 2 and 3 was enhanced
at 5 DPA (128nmol g−1 fresh weight and 81nmol g−1 fresh weight,
respectively) and further at 10 DPA (378nmol g−1 fresh weight and
300nmol g−1 fresh weight, respectively) (Extended Data Fig. 10c–e).
Trehalose levels were also higher (30–70 nmol g−1 fresh weight
compared to endogenous levels of 13nmol g−1 fresh weight), consistent
with the metabolism observed in A. thaliana.
Next, the effects of signalling precursors on plant resilience and
recovery were analysed. Drought is still the biggest global factor limiting
crop yields, even in developed countries29. When 4-week-old wheat
plants were sprayed with compounds 2 or 3 (30ml, 1mM, once) after 9
days of drought, the regrowth effects following resumption of watering
1 day after treatment were substantial (Fig. 6a, b). Regrowth of new
tissue from plants cut back after drought was also higher in precursor-
treated plants (Fig. 6c, d). This demonstrated both growth of new tissue
(resurrection response) and salvage and growth of existing tissue
(recovery response). T6P solution alone gave identical results to water
(Fig. 6), consistent with the inability of T6P to enter directly into plants,
further highlighting the design principles of signalling precursors.
In conclusion, we have shown here that a chemical strategy can
directly control amounts of an important sugar signalling molecule
in vivo. The collected data are consistent with the signalling action
of released T6P. For example, the mass balance of added signalling
precursor appears insufficient to simply act as a carbon source. That
said, we do not discount other possible mechanisms behind the
noteworthy traits that we have observed here. The apparent result of
‘biosynthetic amplification’ observed from signalling precursors is, we
believe, a promising concept; we calculate here up to 50-fold ‘molecular
amplification’ of the plant sugar product compared to the precursor.
It may therefore be possible to design a self-sustaining production
strategy in which a fraction of the additional starch generated by this
amplification is used as a feedstock chemical for eventual synthesis of
the signalling precursors themselves (Supplementary Discussion and
Supplementary Table 18).
15. Clarke, E. D. & Delaney, J. S. Physical and molecular properties of
agrochemicals: an analysis of screen inputs, hits, leads, and products. Chimia
57, 731–734 (2003).
16. Kirkwood, R. C. Target Sites for Herbicide Action. (Springer, 1991).
17. Delatte, T. L. et al. Determination of trehalose-6-phosphate in Arabidopsis
seedlings by successive extractions followed by anion exchange
chromatography-mass spectrometry. Anal. Biochem. 389, 12–17 (2009).
18. Bjarnholt, N., Li, B., D’Alvise, J. & Janfelt, C. Mass spectrometry imaging of plant
metabolites–principles and possibilities. Nat. Prod. Rep. 31, 818–837 (2014).
19. Cha, S. et al. Direct profiling and imaging of plant metabolites in intact tissues
by using colloidal graphite-assisted laser desorption ionization mass
spectrometry. Plant J. 55, 348–360 (2008).
20. Cheng, J. & Winograd, N. Depth profiling of peptide films with TOF-SIMS
and a C60 probe. Anal. Chem. 77, 3651–3659 (2005).
21. Nunes, C. et al. Regulation of growth by the trehalose pathway: relationship
to temperature and sucrose. Plant Signal. Behav. 8, e26626 (2013).
22. Szecowka, M. et al. Metabolic fluxes in an illuminated Arabidopsis rosette.
Plant Cell 25, 694–714 (2013).
23. Schluepmann, H. et al. Trehalose mediated growth inhibition of Arabidopsis
seedlings is due to trehalose-6-phosphate accumulation. Plant Physiol. 135,
879–890 (2004).
24. Smith, A. M. & Zeeman, S. C. Quantification of starch in plant tissues.
Nat. Protocols 1, 1342–1345 (2006).
25. Givens, R. S., Weber, J. F., Jung, A. H. & Park, C. H. New photoprotecting groups:
desyl and p-hydroxyphenacyl phosphate and carboxylate esters. Methods
Enzymol. 291, 1–29 (1998).
26. Kolbe, A. et al. Trehalose 6-phosphate regulates starch synthesis via
posttranslational redox activation of ADP-glucose pyrophosphorylase.
Proc. Natl Acad. Sci. USA 102, 11118–11123 (2005).
27. Hädrich, N. et al. Mutagenesis of cysteine 81 prevents dimerization of the
APS1 subunit of ADP-glucose pyrophosphorylase and alters diurnal starch
turnover in Arabidopsis thaliana leaves. Plant J. 70, 231–242 (2012).
28. Pellny, T. K. et al. Genetic modification of photosynthesis with E. coli genes for
trehalose synthesis. Plant Biotechnol. J. 2, 71–82 (2004).
29. Boyer, J. S. Plant productivity and environment. Science 218, 443–448 (1982).
30. Zhang, Y. et al. Inhibition of SNF1-related protein kinase1 activity and
regulation of metabolic pathways by trehalose-6-phosphate. Plant Physiol. 149,
1860–1871 (2009).
We speculate that this chemical approach also offers more temporal
and strategic flexibility than genetic methods (for example, a ‘pulse’
to circumvent adaptation effects or in manipulating more genetically
complex crops) as well as the prospect of providing an immediate boost
to productivity at critical times in the plant life cycle (for example, to
allow synchronicity with the sun or to rescue drought-stricken regions,
Supplementary Discussion)— the potential to contribute towards
global food security seems notable and immediate. Given the wide-
spread importance of cell signalling and of carbohydrates in biology,
this system may also have even wider utility.
31. Martins, M. C. M. et al. Feedback inhibition of starch degradation in Arabidopsis
leaves mediated by trehalose 6-phosphate. Plant Physiol. 163, 1142–1163
(2013).
39. Billington, D. C. Recent developments in the synthesis of myo-inositol
phosphates. Chem. Soc. Rev. 18, 83–122 (1989).
Online Content Methods, along with any additional Extended Data display items and
Source Data, are available in the online version of the paper; references unique to
these sections appear only in the online paper.
Supplementary Information is available in the online version of the paper.
Acknowledgements We thank the BBSRC Selective Chemical Intervention
in Biological Systems initiative (grant reference BB/D006112/1), the
BBSRC Sparking Impact initiative and ICL Innovations for funding.
We thank R. H. Bromilow, S. Powers, E. Tobolkina, for advice and J. Wickens
for technical assistance. NiCE-MSI is supported by the 3D NanoSIMS and
AIMS-HIGHER projects of the Chemical and Biological programme of the
National Measurement System of the UK Department of Business, Innovation
and Skills. B.G.D. was a Royal Society Wolfson Research Merit Award recipient
during the period of research. Rothamsted Research receives strategic
funding from the BBSRC.
Received 2 April 2015; accepted 28 October 2016.
Published online 14 December 2016.
1. Ray, D. K., Mueller, N. D., West, P. C. & Foley, J. A. Yield trends are insufficient
to double global crop production by 2050. PLoS One 8, e66428
(2013).
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of a global crop yield slowdown from climate trends in the next two decades.
Environ. Res. Lett. 9, 074003 (2014).
Author Contributions C.A.G., R.S. and Y.G. are joint first authors. C.A.G., R.S.,
Y.G. and L.F.P. performed experiments. R.S., Y.G. and M.K.Pat. synthesized
compounds. Y.G. and M.K.Pas., I.S.G., R.T.S., J.B. and B.G.D. performed
and/or analysed the mass spectrometry imaging. Y.G. and B.G.D. performed
and/or analysed the tandem mass spectrometry. C.A.G., R.S., Y.G., L.F.P.,
M.J.P. and B.G.D. designed and analysed the experiments. M.J.P. and B.G.D.
wrote the paper. All authors read and commented on the paper.
3. Shewry, P. R. Wheat. J. Exp. Bot. 60, 1537–1553 (2009).
4. Nuccio, M. L. et al. Expression of trehalose-6-phosphate phosphatase in maize
ears improves yield in well-watered and drought conditions. Nat. Biotechnol.
33, 862–869 (2015).
5. Figueroa, C. M. & Lunn, J. E. A tale of two sugars: trehalose 6-phosphate and
sucrose. Plant Physiol. 172, 7–27 (2016).
6. Mayer, G. & Heckel, A. Biologically active molecules with a ‘light switch’. Angew.
Chem. Int. Edn Engl. 45, 4900–4921 (2006).
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare competing financial interests:
details are available in the online version of the paper. Readers are welcome to
comment on the online version of the paper. Correspondence and requests for
materials should be addressed to M.J.P. (matthew.paul@rothamsted.ac.uk) and
B.G.D. (Ben.Davis@chem.ox.ac.uk).
7. Adams, S. R. & Tsien, R. Y. Controlling cell chemistry with caged compounds.
Annu. Rev. Physiol. 55, 755–784 (1993).
8. Givens, R. S. & Kueper, L. W. Photochemistry of phosphate-esters. Chem. Rev.
93, 55–66 (1993).
9. Ellis-Davies, G. C. Caged compounds: photorelease technology for control
of cellular chemistry and physiology. Nat. Methods 4, 619–628 (2007).
0 0 M O N T H 2 0 1 6
| V O L 0 0 0 | N A T U R E | 5
© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

RESEARCH LETTER
METHODS
acid in H2O) and solvent B (1.0% formic acid in H2O:CH3CN (75:25)), were used
Synthesis of signalling-precursor compounds 1–4. 1H-tetrazole solution as the mobile phase at a flow rate of 0.4ml min−1. For the Xevo G2-S QTof MS,
(0.45 M in CH3CN) (0.6 ml, 0.24 mmol, 2.0 equiv.) was added into a stirred the electrospray source was operated with a capillary voltage of 2.0kV and a cone
solution of compound 12 (100mg, 0.12mmol, 1 equiv.) and bis-(2-nitrobenzyl)-N, voltage of 30V. Nitrogen was used as the desolvation gas at a total flow of 800l h−1
.
N-diisopropylphosphoramidite (compound 9; 78.3mg, 0.18mmol, 1.5 equiv.) in The intact molecular ion of T6P was detected as m/z 421.0759 (C12H22O14P,
anhydrous CH2Cl2 (5ml) under an argon atmosphere at 0°C. The resulting reaction calculated as 421.0753) in a negative ion mode. The time of flight (ToF) tandem
mixture was stirred at 0–5°C and progress of the reaction was monitored by thin- mass spectrum of the parent ion 421.00 was then obtained in a negative ion mode
layer chromatography (petroleum ether:ether, 8:2) and mass spectrometry. After for the m/z range from 50 to 500 using optimized collision energy of 20 eV. For
complete disappearance of starting material (1h), tBuOOH (0.1ml) was added the Quattro micro API MS, the electrospray source was operated with a capillary
at 0°C and stirring was continued for another 30min. After 30min the reaction voltage of 3.0kV and a cone voltage of 40V. The quadrupole tandem mass spectra
mixture was concentrated in vacuo and the residue was suspended in methanol of the parent ion 421.0 of T6P and S6P were obtained in a negative ion-mode for
(2ml) and stirred in the presence of 30mg of Dowex-H+ resin for 1h at room the m/z range from 50 to 500 using collision energy of 20eV. With the reference
temperature to globally remove trimethylsilyl groups. Dowex-H+ was removed to standard fragmentation patterns, tracking of the fragment ions of T6P in
through filtration and the filtrate was concentrated, which on flash chromatog- the plant sample was also performed by quadrupole tandem mass spectra. The five
raphy (water:isopropanol:ethyl acetate, 1:2:8) purification yielded compound 1 most intense m/z peaks recorded in the MS/MS spectrum of T6P; m/z 78.3, 96.4,
(70 mg) in 87% isolable yield. Similar reaction protocols were used for the 138.6, 240.9 and 421.0 (unfragmented) were also selected for multiple reaction
synthesis of compounds 2 and 3. Compound 4 was obtained when a stirred monitoring (MRM) and cross-referenced with selected ion recordings (SIR) for
solution of 12 (100mg, 0.12mmol) in pyridine (2 ml) at room temperature was the intact molecular ion m/z 421.0.
treated with POCl3 (0.012ml, 0.132mmol) for 10min followed by addition of
For seedling liquid culture, seeds of A. thaliana were grown in liquid culture as
4,5-dimethoxy-2-nitrobenzyl alcohol (76.7mg, 0.36mmol) and continuous stirring described previously21. Once the seedlings were 7 days old, oNPE-T6P (compound 3)
for 1h. The resulting reaction mixture was concentrated in vacuo to yield a crude or DMNB-T6P (compound 2) were added to the growth medium to a final
product mixture, which was treated with Dowex-H+ (30mg) in methanol (2ml). concentration of 1 mM. Plants were left under growth lights to take up the
After filtration, concentration in vacuo and flash chromatography purification compounds for 24h. To facilitate precursor release, plants were placed under
yielded compound 4 (45mg, 62%) as a pure sticky solid. For additional details see 23μmol m−2 s−1 UV for 2h, after which they were returned to previous envi-
Supplementary Methods.
ronmental conditions. Samples were taken for analysis before addition of the
In planta uptake of signalling-precursor compounds and release of trehalose- compound, 1 day after addition, after 30, 60 and 120min during UV treatment, and
6-phosphate and metabolites. In planta uptake was carried out using A. thaliana sampled again at 1 and 2 d after UV treatment. Samples were weighed, snap-frozen
plantlets. A. thaliana (Columbia 0) seeds were surface-sterilized for 10min in 10% and stored at −80°C.
sodium hypochlorite, 0.01% Triton X-100 and then copiously washed with sterile
For enzymatic sugar analysis, sugars were extracted from 5–10mg of A. thaliana
water and stratified for 3 d at 4°C. Seeds were sown onto 0.5ml solid medium (0.5× ground under liquid nitrogen, 1ml of 80% was added and the sample was heated
Murashige and Skoog medium with Gamborg’s vitamins (Sigma P0404), 0.5% at 100°C for 1h, samples were centrifuged for 10min at 13,000g to remove debris.
sucrose and 0.5% agar) in 0.5 ml Eppendorf tubes, pierced in the bottom with a tiny The samples were added to assay buffer24. Enzymatic reactions were performed
hole. The tubes were arrayed in hand-cut polystyrene racks in Phyta trays (Sigma) as described previously32 using hexokinase, glucose-6-phosphate dehydrogenase,
and floated on liquid medium (same as solid medium but lacking sucrose and phosphoglucose isomerase and invertase from Sigma-Aldrich (H4502, G8404,
agar). Plantlets were grown under the following conditions: 12h day under Philips P5381 and I9274, respectively). Two technical replicates were carried out for each
master TL-D 840/58W fluorescent lights outputting 250μmol m−2 s−1, with 23°C sample, a total of three biological replicates were analysed. See Supplementary
day and 18°C night temperatures. At 18 days after sowing the liquid medium was Methods for further details.
removed and the tubes were sealed with electrician’s tape. All plants were topped Extraction and measurement of starch in planta. Three or four chemical- and
up with 0.5× Murashige and Skoog medium with no sucrose.
UV-light-treated plantlets were pooled and weighed (fresh weight, 70–100mg) for
Plants were treated with compounds by adding 10μl of a 50mM stock prepared each biological replicate. Extraction was based on literature methods24. Samples
in water or 1% DMSO to the agar medium, avoiding contact with aerial parts. were ground in liquid nitrogen to a fine powder in a mortar. The powder was
The final concentration of the precursor compound in the agar medium was rapidly extracted with 1ml 80% ethanol at 80°C, followed by 2×0.5ml to rinse,
1mM. After a certain period of time (after 24h, 48h and 72h) the aerial part was samples were then transferred to a 2-ml eppendorf at 100°C and heated for
harvested carefully, weighed and extracted in H2O:MeOH (1:1) under liquid 2–3min until just boiling. Tubes were transferred to a water bath at 80°C while
nitrogen. The crude fresh plant extract thus obtained was analysed by mass other samples were accumulated. Samples were centrifuged at 13,000g for 10min
spectrometry and HPLC.
to collect all solid material. The pellet was extracted twice more with 2ml, 80%
For in planta T6P release experiments, compound-treated plants were exposed hot ethanol. The pellet was washed with 1 ml water, the supernatant removed
to UV-light treatment after 72h. UV treatments consisted of: (a) 8-h exposure to and 100μl water added. The pellet was homogenized to a smooth consistency
natural daylight; (b) 8-h exposure to a 100 W UV spotlight (BlackRay B-100AP) at with an Eppendorf micropestle before being made up to final volume of 500μl
a distance of 18cm; (c) 8-h exposure to an 8 W UV bulb (365nm, Gelman transil- with water. Samples were heated at 100°C for 10min to gelatinize starch granules.
luminator Model 51438) at a distance of 6cm; or (d) exposure for two 8-h periods Duplicate aliquots (100μl) were removed and digested with α-amylase (2U) and
to 8 W. UV treatments were in addition to normal growth lights. Control plants amyloglucosidase (6U) in 0.05M sodium acetate pH 4.8 for 4h at 37°C. Control
(except for daylight treatment) were treated under the same conditions but without digests without enzyme were also set up. Glucose released from digested starch
UV light. At the end of the day of exposure to UV or visible light, the aerial parts of was measured using an enzymatic assay coupled to the reduction of NADP to
the plants were quickly harvested, weighed and frozen in liquid nitrogen. For starch NADPH33 and adapted for a microtitre plate reader. 10–20μl of digest was assayed
extractions, a moderate light regime was selected of a single 8h exposure to 8 W in triplicate. Starch content is expressed as hexose equivalents per gram fresh
UV light. After irradiation plants were returned to the growth room for a further weight. See Supplementary Methods for further details.
day (day 5) to recover after light treatment and to respond to altered T6P levels SnRK1 activity. Kinase activities were determined by measuring the incorporation
before being harvested as above. Frozen tissue was stored at −80°C until extracted. of radiolabelled phosphate into the ‘AMARA’ peptide (AMARAASAAALARRR;
Harvested plant material was extracted by liquid/liquid extraction (LLE) Biomol International) substrate and were carried out as described previously30
.
followed by solid phase extraction (SPE) for T6P analysis17. For LLE/SPE ADP-glucose pyrophosphorylase activity. Enzyme activity was measured as
extractions around 25 mg plant tissue was used, pooled from several plants. described previously34
.
Samples were reconstituted in 50μl of H2O:MeOH (1:1) and 10μl was used for Application of precursors to wheat. Spring wheat (T. aestivum Cadenza) seeds
T6P analysis; T6P release was determined using quantitative HPLC–MS (Quattro, were sown in Rothamsted standard compost mix and grown in controlled
Waters), with 2-deoxy-glucose-6-phosphate as a calibration internal standard.
environment conditions with a photoperiod of 16 h light, 8 h dark, day/night
Liquid chromatography tandem mass spectrometry (LC–MS/MS) was used temperatures of 20°C/16°C, photon-flux density of 600μmol m−2 s−1, and ambient
to confirm the identity of disaccharide monophosphates via fragmentation relative humidity. Once the plants had reached anthesis, solutions of oNPE-T6P
pattern analysis performed on a Waters Xevo G2-S QTof (quadrupole time- (compound 3) and DMNB-T6P (compound 2) (0.1, 1 or 10mM) as well as control
of-flight) mass spectrometer coupled to a Waters Acquity Ultra Performance Liquid solutions, water, 1mM T6P or 1mM trehalose were made up in distilled water with
Chromatography (UPLC) system, and a Waters Micromass Quattro micro API 0.1% TWEEN-20. At 5, 10, 15 and 20 days after anthesis, either the ears, or the
Mass Spectrometer coupled to a Waters 1525μ binary HPLC pump and a Waters whole plant were sprayed individually with 5 or 50ml of precursor, respectively.
2777 auto sampler using a SIELC Primesep SB column. Solvent A (0.1% formic Leaf samples were taken at 5, 10, 15, 20 and 25 days after anthesis for chlorophyll
© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

LETTER RESEARCH
content analysis, grain was harvested at maturity for analysis. Chlorophyll content which they remained under the previously stated growth conditions. After 24h,
of leaves was measured by methanol extraction and spectrophotometry35. Starch the plants were exposed to UV light at 23μmol m−2 s−1 for 2h to facilitate T6P
content of grain was measured enzymatically24and protein content was measured release. Plants were left for 2h to recover, frozen and dehydrated in a vacuum
by Bradford’s assay36
.
chamber before mass spectometry imaging analysis. Reference materials were
For the drought treatment, vegetative Cadenza wheat plants were grown in drop-dried on clean substrates. The ToF–SIMS mass spectrometry imaging analysis
the same compost and environments as above. Once the plants had reached was performed with the ToF-SIMS IV mass spectrometer (IONTOF, Muenster,
Feekes stage 4, water was withheld for 10 days. On day 9, 30-ml, 1mM solutions Germany) from three leaves, a control, and one leaf each treated with compounds
of oNPE-T6P (compound 3) and DMNB-T6P (compound 2) were applied to all 2 or 3. A pulsed 25-keV Bi3+ primary ion source was used as the analysis beam
above-ground biomass, on day 10 the watering schedule was reinstated. Plants were (pulse width, 23ns, mass resolution, (m/Δm), 5,000). Mass spectra of the reference
harvested to measure biomass production every 5 days for 30 days after rewatering. material were obtained in positive and negative ion mode at a primary ion dose
Both experiments were completed in biological replicates of six.
of 1.1×1011 ions cm−2. The leaf ion images were also collected in both polarities
For quantification of T6P, trehalose and sucrose in wheat samples, the harvested with a dose of 5.4×1010 ions cm−2. An electron flood gun was employed for charge
wheat grains were weighed, snap-frozen and stored at −80°C. Wheat grain was compensation during the data acquisition. Mass spectrometry data was analysed in
ground to a fine powder in liquid nitrogen and the sugars were extracted by LLE for ION-TOF SurfaceLab 6.4 software and further processed in MATLAB and Origin
T6P, trehalose and sucrose analysis using the same LC–MS quantification method Pro. Known melissic acid markers19 (m/z 435.4, C30H59O, [M−OH]− and 451.4
as for A. thaliana.
C30H59O2, [M−H]−) were used. See Supplementary Methods for further details.
MALDIMS imaging data were acquired using identically prepared leaf
For minimal spray application, spring wheat (T. aestivum Cadenza) seeds were
sown in Rothamsted standard compost mix and grown in controlled environment samples with a modified QSTAR XL Qq-ToF instrument (Sciex, Ontario, Canada)
conditions with a photoperiod of 16h light, 8h dark, day/night temperatures of fitted with a Nd:YAG laser (Elforlight Ltd, Daventry, UK) operated at 1,000kHz
20°C/16°C, photon-flux density of 600μmol m−2 s−1 and ambient relative humidity. in positive ion mode with a fluence of approximately 205J m−2 and a pixel size of
Once the plants had reached anthesis, solutions of compounds 2 or 3 (1mM and 200μm×200μm. The QSTAR was operated in continuous raster sampling mode.
10mM) and a water control, were made up in distilled water with 0.1% TWEEN- The sample was affixed to a stainless steel target plate with double-sided tape and
20. At 10 days after anthesis, the top 20cm of above ground biomass encompassing sprayed with CHCA (5mg ml−1 CHCA in 80% methanol, 0.1% TFA) using auto-
ears and flag leaves were sprayed individually with 25ml of the compounds. Grain mated spray deposition (TM Sprayer, HTX Technologies, Carrboro, USA). Data
from individual ears was harvested at maturity for analysis. All wheat experiments were converted from the proprietary .wiff format into .mzML using AB MS Data
were repeated twice, with 3 technical and 6 biological replicates completed at each Converter version 1.3 (Sciex). These mzML files were then converted to .imzML
stage of analysis.
using imzMLConverter38 and processed in custom-made MATLAB software
RNA extraction, cDNA synthesis and qRT–PCR. Total RNA was extracted from (version R2014b, Math Works Inc, USA). Images are created by summing across
50mg snap-frozen leaf tissue from A. thaliana Columbia using the Ribopure Kit the full-width, half-maximum of the peak of interest to give the intensity within
(Ambion) according to the manufacturer’s instructions. RNA was quantified using a each corresponding pixel. See Supplementary Methods for further details.
Nanodrop spectrophotometer and integrity of RNA was visualized using denaturing Statistical methods. ANOVA was applied to data to test for differences between
agarose gel electrophoresis37. DNA was removed using RQ1 RNase-free DNase treatments. A natural log transformation was used where necessary to ensure
(Promega). cDNA was synthesized using SuperScript III First-Strand Synthesis constant variance. The GENSTAT statistical system was used for this analysis.
System (ThermoFisher Scientific) using 2μg of total RNA and oligo-dT primers Data availability. Primary data for Figs 2, 4, 5 and 6 are provided as spreadsheets.
according to the manufacturer’s instructions. Gene expression was quantified using All other data are available on request.
SYBR Green chemistry on a Real-Time PCR system 7500 (Applied Biosystems).
32. Spackman, V. M. T. & Cobb, A. H. An enzyme-based method for the rapid
Total reaction size was 20μl, containing 10μl SYBR Green Jumpstart Taq ReadyMix
determination of sucrose, glucose and fructose in sugar beet roots and the
effects of impact damage and postharvest storage in clamps. J. Sci. Food Agric.
82, 80–86 (2002).
(Sigma Aldrich), 2μl cDNA and 0.5mM primers. PCR used an initial denaturation
stage of 95°C for 2min, followed by 40 cycles of 95°C for 15s, 60°C for 1min. The
specificity of products was confirmed by performing a temperature gradient analysis
of products at temperatures ranging from 55°C to 95°C with 0.5°C increments.
Two technical replicates were completed for each sample, a total of three biological
replicates were analysed. Relative quantification of gene expression was performed
using the Livak method using ubiquitin-transferase family protein as the reference
gene. Primers used for SnRK1 marker gene expression, and starch gene expression
are listed in Supplementary Tables 19 and 20, respectively.
Mass spectometry imaging methods. A. thaliana used in ToF–SIMS (time-
of-flight secondary ion mass spectrometry) imaging were grown in Petri dishes on
0.5× Murashige and Skoog medium with 0.8% agar for 10 d, with a photoperiod of
16h light, 8h dark, day/night temperatures of 23°C/18°C and photon-flux density
of 250μmol m−2 s−1. Plants were then transferred to Petri dishes containing the
same media supplemented with 1mM of either compound 2 or 3 for 24h, during
33. Jones, M. G. K., Outlaw, W. H. & Lowry, O. H. Enzymic assay of 10 to 10 moles
of sucrose in plant tissues. Plant Physiol. 60, 379–383 (1977).
34. McKibbin, R. S. et al. Production of high-starch, low-glucose potatoes through
over-expression of the metabolic regulator SnRK1. Plant Biotechnol. J. 4,
409–418 (2006).
35. Ritchie, R. J. Consistent sets of spectrophotometric chlorophyll equations for
acetone, methanol and ethanol solvents. Photosynth. Res. 89, 27–41 (2006).
36. Bradford, M. M. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein–dye binding.
Anal. Biochem. 72, 248–254 (1976).
37. Sambrook, J. & Russell, D. Molecular Cloning: A Laboratory Manual, 3rd Edn
(Cold Spring Harbor Laboratory Press, 2000).
38. Race, A. M., Styles, I. B. & Bunch, J. Inclusive sharing of mass spectrometry
imaging data requires a converter for all. J. Proteomics 75, 5111–5112
(2012).
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RESEARCH LETTER
Extended Data Figure 1 | The central role of T6P in plants and design of
a chemical strategy for the control of T6P production. a, Photosynthesis
generates sucrose, which is translocated to growing regions of the plant.
Inside the cell, a pool of core metabolites are substrates for biosynthetic
processes that determine growth and productivity. T6P is synthesized
from UDPG and G6P by trehalose 6-phosphate synthase (TPS) and
therefore reflects the abundance of sucrose. It is broken down by trehalose
phosphate phosphatase (TPP). Increasing T6P stimulates starch synthesis
and inhibits SnRK1, a protein kinase central to energy conservation and
survival during energy deprivation. Inhibition of SnRK1 by T6P thus
diverts carbon skeleton consumption into biosynthetic processes.
b, The trehalose biosynthetic pathway. c, T6P is plant-impermeable.
Plant-permeable variants allowed subsequent photo-activated release.
d, Generalized mechanism of light-activated release of precursors.
e, Release of T6P by light irradiation from signalling precursors 1–4
in vitro. 31P nuclear magnetic resonance spectroscopy at different
time points of light irradiation confirming the activation of signalling
precursors (1–4) and release of T6P. Time points for compound 1: 0, 30,
60, 150 and 360 min; for compound 2: 0, 60, 120, 300, 420 and 600 min;
for compound 3: 0, 15, 30, 45 and 60 min; and for compound 4: 0, 60, 120,
240, 360 and 420 min. f, g, 1H (f) and 31P (g) nuclear magnetic resonance
spectra, after complete photolysis of the signalling precursor confirming
the release of T6P.
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LETTER RESEARCH
Extended Data Figure 2 | Inhibition of SnRK1. Signalling precursors
(1–4), T6P released from 1–4 (r1, r2, r3, r4) and T6P standard (T6P)
were tested for inhibition of SnRK1 activity. T6P (0.26 mM) inhibits
SnRK1 activity to approximately 36% of the original activity. Signalling-
precursor compounds show no such inhibition, whereas UV-released
compounds show the same inhibition as free T6P. SnRK1 activity was
determined by the level of incorporation of phosphate into a peptide
substrate (min−1 mg−1 protein). (Data are shown as mean s.e.m.; n =3).
The activities of assays treated with precursors or released T6P were not
significantly different from their controls (P <0.001, LSD) using one-way
ANOVA of data transformed with a natural log scale.
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or [M −H]−, of pure signalling precursors 1–4. d, HPLC (left) and mass
spectometry (right) data, [M +Na]+ or [M −H]−, of plant samples after
treatment with signalling precursors 1–4. In 3, the partially uncaged
molecule also accumulated and was detected (coloured light blue).
Extended Data Figure 3 | In planta uptake analysis of signalling
precursors 1–4. a, Schematic of protocol used for uptake analysis.
b, Calibration curves for oNB-T6P (1), DMNB-T6P (2), oNPE-T6P (3)
and mono-DMNB-T6P (4), respectively. Data are shown as mean s.e.m.;
(n =2). c, HPLC (left) and mass spectrometry (right) data, [M +Na]+
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Extended Data Figure 4 | See next page for caption.
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Extended Data Figure 4 | Extraction and quantification of T6P.
a, Schematic of the protocol used for preparation of samples for T6P
quantification. LLE, liquid/liquid extraction; SPE, solid phase extraction;
AEC–MS, anion exchange chromatography–mass spectrometry.
b, Liquid chromatograms of T6P, S6P and 2DG6P separation (top)
using conditions optimized in Supplementary Table 8 (entry 7) and the
representative LC–MS chromatograms of extraction samples treated with
signalling precursors (middle) and water control (bottom). c, d, Liquid
chromatograms of different concentrations of T6P (500, 250, 100, 50,
25, 10 or 5 μM) using a constant concentration (100 μM) of 2DG6P as an
internal standard. e, Resulting calibration curves of the T6P peak area and
and S6P (bottom) by quadrupole time-of-fight tandem mass spectrometry
(QToF–MS/MS) in negative ion mode. g, Fragmentation patterns of T6P
(top) and S6P (middle) by triple quadrupole tandem mass spectrometry
(QqQ–MS/MS) in negative ion mode and the T6P fragment ions tracking
in the plant matrix (bottom). h, HPLC chromatograms of T6P/S6P by
selected ion recording (SIR) of the intact molecular ion (m/z 421.0) and
multiple reaction monitoring (MRM) of the fragment ions give the same
retention time for each compound. i, The LC–MS quantification method
through SIR and LC–MS/MS quantification method through MRM of the
T6P level in the DMNB-T6P 2-treated plant sample. From bottom to top:
integration of the T6P trace (2,661) using SIR of m/z 421.0, integration of
T6P/2DG6P ratio against T6P concentrations (in μM) in water as well as in the T6P trace (2,550) using MRM of m/z 78.6, 96.3, 138.7, 241.0 and 421.0,
the plant matrix. f–h, LC–MS/MS analysis of T6P and S6P from samples
of plant treated with compound 2. f, Fragmentation patterns of T6P (top)
integration for each fragment ion m/z 78.6 (801), m/z 96.3 (868), m/z 138.7
(76), m/z 241.0 (404) and m/z 421.0 (392).
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Extended Data Figure 5 | See next page for caption.
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Extended Data Figure 5 | Analysis of A. thaliana plantlets following
treatment. a–c, Phenotype analysis. a, Fresh weight of plantlets versus
concentration of signalling precursors of T6P (1–4) and G6P precursors
(14–17) in medium after three days (72 h) of uptake. Data are shown as
mean s.e.m.; n =3. Each T6P precursor is shown (top) together with
its G6P analogue (bottom). Visual appearance of a typical plantlet was
analysed for a given concentration of precursors at the point of harvest.
b, Phenotype of plants at the end of light treatments. Plants were allowed
to take up compounds for 72 h and were then treated the next day with
light treatments. Light treatments: GL, growth light irradiance
differences were observed between treatments. Water (left),
0.1 mM oNPE-T6P (compound 3) (middle), oNPE-G6P(1-OMe)
(compound 16) (right). Scale, tube diameter =10 mm. d–g, Biosynthetic
effects of increasing T6P in planta. d, Starch level at the end of the day in
UV-treated (8 W, 23 μmol m−2 s−1, 8 h) plants fed with compound 3 +UV
is significantly higher than plants treated with water +UV (n =9, data
are shown as mean s.e.m.). Samples for starch were taken 1 day after
UV treatment. e, ADP-glucose pyrophosphorylase (AGPase) activity is
increased in UV +compound 3 plants compared to compound 3 only,
UV only, water only, UV +water-treated plants and plants treated with
compound 16 (n =3, data are shown as mean s.e.m.). f, Starch synthesis
rate in UV-treated (20 μmol m−2 s−1, 8 h) plants treated with compound 3
(data are shown as mean s.e.m., n =3). g, Starch level at the beginning
(data are shown as mean s.e.m.; n =3) and at the end (data are shown
as mean s.e.m.; n =4) of the day in UV (20 μmol m−2 s−1, 8 h) +water-
treated (solid circles) and UV +compound 3-treated (empty circles)
plants. A. thaliana used in e–g were grown at a light regime of 12 h
day–12 h night, at 250 μmol m−2 s−1, and 23 °C day/18 °C night
temperatures, treated with compounds 18 d after sowing, and exposed
to UV light 72 h after addition of the compound. Asterisks in d–f denote
significance according to ANOVA (P =0.002). Asterisk in g denotes
significance by one-way ANOVA (LSD 5% =11.19).
250 μmol m−2 s−1. UV 8 W and UV 100 W were growth light irradiance
supplemented with UV light (365 nm). Daylight, part sun/part
cloud; irradiance between 250 μmol m−2 s−1 under cloud and
1,440 μmol m−2 s−1 under full sun. Compounds were fed to the
plants to a final concentration of 1 mM. Phenotype of plants fed with
compound 3 at a reduced final concentration of 0.1 mM are shown in
the right-hand panel. Scale, diameter of the plastic tube mouth =10 mm.
c, Typical A. thaliana phenotypes in the starch experiment. Plants were
treated with a final medium concentration of 0.1 mM compound or water
for 72 h and then exposed to 8-h, 8 W UV treatment. The plants were
allowed to recover for another 24 h and were harvested at the end of the
day and the starch content was measured. No significant phenotypic
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Extended Data Figure 6 | Quantification of in planta metabolites.
a, LC–MS chromatograms of trehalose, sucrose, glucose and fructose
separation using a HILIC column, for details see Supplementary
Information. b, Liquid chromatograms and peak areas of different
concentrations of trehalose (100, 50, 25, 10 or 5 μM) and glucose
(500, 250, 100, 50, 25, 10 or 5 μM). c, Calibration curves of the trehalose
peak area against the concentrations (in μM) and glucose peak area
against the concentrations (in μM). d, e, Same as Fig. 4, 7-day-old
A. thaliana seedlings grown in liquid culture were treated with 1 mM of
compounds 3 or 2, control seedlings were treated with water. Seedlings
were left under growth lights to allow for uptake of the signalling
precursors for 24 h, before exposure to 23 μmol m−2 s−1 UV for 2 h.
Measurements were taken 1 day after uptake (pre-UV), 30, 60 and 120 min
after initiation of UV treatment, and 1 and 2 d after initiation of UV
treatment. See Fig. 4 for T6P content, trehalose content, sucrose content,
glucose content; here fructose content (d)and Fresh weight biomass (e)
are shown. *P <0.05); **P <0.01 (Student’s t-test). Data are shown as
mean s.e.m. (n =3).
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Extended Data Figure 7 | Dynamics of 13C-T6P, 13C-trehalose,
13C-glucose and T6P in A. Thaliana treated with 13C-labelled
precursor 2*. 1 mM of DMNB-13C-T6P (2*) was added to the growth
medium of 7-day-old A. thaliana seedlings. The plants were left under
growth light to allow uptake for 24 h and the uncaging was performed
under 23 μmol m−2 s−1 UV for 2 h. Samples were harvested for analysis
at different time points: pre-UV, 30, 60 and 120 min (after onset of
UV irradiation) and 1 and 2 days after UV irradiation. a, Synthesis of
universally 13C-labelled 2* in essentially the same manner as for 2.
b, Amount of 13C-T6P released over time in planta. c, Amount of
13C-trehalose accumulated. d, Amount of 13C-glucose accumulated.
e, Amount of endogenous T6P. f, Overview of 13C tracking of T6P and
metabolites. Data are shown as mean s.e.m.; n =3; *P <0.05; **P <0.01
(Student’s t-test).
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[M +Na]+ or [M −H]−, of pure G6P(1-Ome) precursors 14–17. For
HPLC conditions see the Supplementary Information section 1. d, HPLC
and mass spectometry data, [M +Na]+ or [M −H]−, of uptaken
G6P(1-Ome) precursors14–17 in planta. e, Calibration curves for
compounds 15, 16 and 17, respectively (n =2 in all cases).
Extended Data Figure 8 | Synthesis, in vitro SnRK1 inhibition studies
and in planta uptake analysis of G6P(1-OMe) precursors 14–17.
a, Design and synthesis of oNB-G6P(1-OMe) (14), DMNB-G6P(1-OMe)
(15), oNPE-G6P(1-OMe) (16) and mono-DMNB-G6P(1-OMe) (17).
b, Lack of inhibition of SnRK1 by G6P(1-OMe) analogues. Data are
shown as mean s.e.m., (n =3). c, HPLC and mass spectometry data,
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Extended Data Figure 9 | Transcript abundance of genes involved in
starch synthesis and SnRK1 marker genes in response to caged T6P
precursor application of 7-day-old A. thaliana seedlings in liquid
culture. Seedlings were treated with a final concentration of 1 mM of
compounds 2 or 3, uptake was allowed for 1 d under the growth lights,
before treatment with 23 μmol m−2 s−1 UV light for 2 h to facilitate
uncaging. a, b, Transcript fold change after 60 min of UV treatment (a)
and 1 d after UV treatment (b) of SnRK1 marker genes. Marker genes
normally downregulated by SnRK1: TPS5, UDPGDH (At3g29360) and
bZIP11, and marker genes normally upregulated by SnRK1: TPS8, ΒGAL
and ASN1. c, Transcript fold change of starch synthesis genes after 60 min
of UV treatment. Genes involved in starch synthesis: APL3, SS3, BE1 and
GBSS1. d, Transcript fold change after 60 min of UV treatment for starch
degradation genes. Genes involved in starch degradation: BAM1, BAM3,
BAM4 and GWD3. Changes in transcripts for enzymes of degradation
were more equivocal with GWD3 increasing and BAM genes showing
small changes or decreasing (BAM3). All data were normalized to a
ubiquitin control. Data are shown as mean s.e.m. of three independent
samples.
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Extended Data Figure 10 | Additional effects in wheat. a, Chlorophyll
content of leaves after anthesis of ear treatments. b, Chlorophyll content
of leaves after anthesis of whole-plant treatments. c–e, T6P release and
metabolism in wheat. Developing wheat grain were treated with T6P,
or compounds 2 or 3 (all 1 mM) 5 or 10 days after anthesis (DPA) and
harvested 1 day later. c, Amount of T6P in wheat grains (n =3; data
are shown as mean s.e.m.). d, Trehalose (n =3; data are shown as
mean s.e.m.). e, Sucrose (n =3; data are shown as mean s.e.m.).
*P <0.05; **P <0.01 (Student’s t-test). f, Dose response grain yield per
plant to T6P precursors (0.1, 1 or 10 mM oNPE-T6P (3) or DMNB-T6P (2)
and water, T6P and trehalose controls) sprayed to ears (5 ml) or to the
whole plant (50 ml) at 5, 10, 15 and 20 days after anthesis. g, Grain yield
per ear in response to a single time point spray (5 ml to the ear at 10 days
after anthesis). *P <0.05 (f, g) compared to water control (Student’s t-test).
Data are shown as mean s.e.m. (n =6).
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