Journal of Experimental Botany, Vol. 66, No. 18 pp. 5531–5542, 2015
doi:10.1093/jxb/erv228 Advance Access publication 22 May 2015
RESEARCH PAPER
Simple and robust determination of the activity signature
of key carbohydrate metabolism enzymes for physiological
phenotyping in model and crop plants
Alexandra Jammer1,*, Anna Gasperl1,*, Nora Luschin-Ebengreuth1,*, Elmien Heyneke1,†,
Hyosub Chu1, Elena Cantero-Navarro1,‡, Dominik K. Großkinsky1,§, Alfonso A. Albacete1,‡,
Edith Stabentheiner1, Jürgen Franzaring2, Andreas Fangmeier2, Eric van der Graaff1,§
and Thomas Roitsch1,§,¶,**
1 2 Institute of Plant Sciences, Karl-Franzens-Universität Graz, Schubertstrasse 51, 8010 Graz, Austria
Institute of Landscape and Plant Ecology, University of Hohenheim, August-von-Hartmann-Strasse 3, D-70599 Stuttgart, Germany
* These authors contributed equally to this work.
Present address: Max-Planck-Institut für Molekulare Pflanzenphysiologie, Wissenschaftspark Golm, Am Mühlenberg 1, D-14476
Potsdam, Germany
‡
Present address: Departamento de Nutrición Vegetal, CEBAS-CSIC, Campus Espinardo, Murcia 30100, Spain.
§
Present address: Department of Plant and Environmental Sciences, Copenhagen Plant Science Centre, University of Copenhagen,
Højbakkegård Allé 13, 2630 Taastrup, Denmark.
¶
Present address: Global Change Research Centre, Czech Globe AS CR, v.v.i., Drásov 470, Cz-664 24 Drásov, Czech Republic.
** To whom correspondence should be addressed. E-mail: [email protected]
†
Received 24 March 2015; Revised 14 April 2015; Accepted 21 April 2015
Editor: Roland Pieruschka
Abstract
The analysis of physiological parameters is important to understand the link between plant phenotypes and their genetic
bases, and therefore is needed as an important element in the analysis of model and crop plants. The activities of enzymes
involved in primary carbohydrate metabolism have been shown to be strongly associated with growth performance, crop
yield, and quality, as well as stress responses. A simple, fast, and cost-effective method to determine activities for 13
key enzymes involved in carbohydrate metabolism has been established, mainly based on coupled spectrophotometric
kinetic assays. The comparison of extraction buffers and requirement for dialysis of crude protein extracts resulted in a
universal protein extraction protocol, suitable for the preparation of protein extracts from different organs of various species. Individual published kinetic activity assays were optimized and adapted for a semi-high-throughput 96-well assay
format. These assays proved to be robust and are thus suitable for physiological phenotyping, enabling the characterization and diagnosis of the physiological state. The potential of the determination of distinct enzyme activity signatures as
part of a physiological fingerprint was shown for various organs and tissues from three monocot and five dicot model
and crop species, including two case studies with external stimuli. Differential and specific enzyme activity signatures
are apparent during inflorescence development and upon in vitro cold treatment of young inflorescences in the monocot ryegrass, related to conditions for doubled haploid formation. Likewise, treatment of dicot spring oilseed rape with
elevated CO2 concentration resulted in distinct patterns of enzyme activity responses in leaves.
Key words: Carbohydrate metabolism, dialysis, enzyme activities, kinetic assay, physiological phenotyping, physiological state,
protein extraction, signatures.
© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved.
For permissions, please email: [email protected]
5532 | Jammer et al.
Introduction
The availability of various cost-efficient genomic tools has
revolutionized plant breeding and biotechnology, which was
accompanied by great advancements in non-invasive techniques for plant phenotyping. However, the phenotypic plasticity in response to the variable environment and agricultural
management is ultimately mediated through a highly flexible
and complex metabolism. Thus a holistic, multidimensional
phenomics approach is required to understand the adaptation of crop plants to variable abiotic and biotic factors.
Understanding plant physiological responses is essential to
reveal the link between phenotypes and their genetic bases
(Yin et al., 2004) within a variable environment and agricultural management practice, and to assess the dynamic
temporal responses to external fluctuations (Schurr et al.,
2006). Therefore, a detailed physiological characterization
is required as an integral element for a system biology phenomics analysis of crop yield and quality, as predictive markers in breeding for improvements in agricultural production.
Carbohydrates function not only as the main energy and
carbon source, but also as signalling factors (Halford et al.,
2011) that can interact with hormone signalling (Eveland and
Jackson, 2012). Consequently, the proper regulation of carbohydrate production, distribution, and allocation is essential for plant development and stress responses. Significant
changes in mRNA expression levels and activities of enzymes
involved in carbohydrate metabolism take place during plant
development, differentiating carbohydrate source and sink
tissues, which are crucial in determining the final biomass and
hence crop yield and quality (Appeldorn et al., 1997; Roitsch,
1999; Calenge et al., 2006; Godt and Roitsch, 2006; Zhu et al.,
2007). Source tissues produce an excess of assimilates, which
are either stored or transported to different sink tissues such
as growing leaves, fruits, and storage organs in most species in
the form of sucrose. Carbohydrate partitioning is determined
by the relative sink strength of the competing sink organs,
but is also affected by abiotic and biotic stress factors (Yang
et al., 2004; Biemelt and Sonnewald, 2006; Berger et al., 2007;
Albacete et al., 2011; Xiang et al., 2011).
Enzyme activities result from the integration of a suite of
transcriptional, post-transcriptional, and post-translational
regulatory mechanisms and thus are ultimately the key determinants of the physiological state of a plant. Invertase (Inv;
EC 3.2.1.26) enzyme activities are strongly associated with
growth characteristics determining plant biomass, fertility,
fruit quality, and senescence (Goetz et al., 2001; Balibrea
Lara et al., 2004; Fridman et al., 2004; Roitsch and Gonzalez,
2004; Sergeeva et al., 2006; Barratt et al., 2009). Functional
analysis showed that enzyme activities for fructokinase (FK;
EC 2.7.1.4) (German et al., 2003), hexokinase (HXK; EC
2.7.1.1) (Dai et al., 1999; Schwartzberg et al., 2011), (fructose
1,6-bisphosphate) aldolase (Ald; EC 4.1.2.13) (Uematsu et al.,
2012), phosphoglucomutase (PGM; EC 5.4.2.2) (Egli et al.,
2010), phosphoglucoisomerase (PGI; EC 5.3.1.9) (Yu et al.,
2000), glucose-6-phosphate dehydrogenase (G6PDH; EC
1.1.1.49) (Esposito et al., 2005), sucrose synthase (Susy; EC
2.4.1.13) (Xu et al., 2012), UDP-glucose pyrophosphorylase
(UGPase; EC 2.7.7.9) (Park et al., 2010), and ADP-glucose
pyrophosphorylase (AGPase; EC 2.7.7.27) (Vigeolas et al.,
2004) are also important for plant growth and development.
Phosphofructokinase (PFK; EC 2.7.1.11) is the key regulator
of carbon flux through glycolysis. Nevertheless, its relevance
for plant growth is less clear (Thomas et al., 1997; MartinezEsteso et al., 2010).
Gene expression levels can be analysed in a relatively
straightforward way using northern blot and real-time PCR
analysis, while microarray and RNA sequencing technologies
enable genome-wide expression analysis. However, quite often
no direct correlation between the mRNA and protein levels
can be found, due to the rate of protein translation, the stability/degradation of proteins, and the regulation of enzyme
activity by substrates, products, protein interactions, and
protein modifications. A comprehensive in situ enzyme activity staining method was published for enzymes involved in
carbohydrate metabolism (Sergeeva and Vreugdenhil, 2002),
which is important for revealing the spatial distribution of
these activities, but does not allow for quantitative measurements. The quantitative determination of enzyme activities in
crude protein extracts by coupled spectrophotometric assays
is widespread. For many key enzymes involved in carbohydrate metabolism, such assays have been established, typically
based on NAD formation or consumption. However, different individual extraction protocols were developed in combination with the various enzyme activity assays. Therefore, a
comprehensive analysis for multiple enzymes requires a high
amount of plant material and excessive labour time. A robotbased platform employing stopped assays coupled to secondary cycling reactions was established (Gibon et al., 2004),
which now comprises various enzymes of carbohydrate and
nitrogen metabolism (Sulpice et al., 2010). However, these
assays are based on end-point measurements rather than
kinetic determination, and require a complex and cost-intensive robot-based experimental platform.
In the present study, the establishment of a universal protein extraction and fractionation method that can be applied
to a wide variety of model and crop plant species and organs
to obtain protein extracts for the determination of activities for key enzymes involved in carbohydrate metabolism
is described. The published spectrophotometric assays for
13 different key enzymes of primary carbohydrate metabolism were miniaturized and adapted for the use in a semihigh-throughput 96-well assay format. The uniform protein
extraction protocol and the robust enzyme activity assays
proved to be suitable for routine physiological phenotyping
of different tissues of various monocotyledonous and dicotyledonous plants, enabling characterization and diagnosis of a
physiological fingerprint for various plant species, including
several crop species. Together with the earlier described analysis of hormone signatures (Großkinsky et al., 2014), these
enzyme activity signatures are important parts of a physiological fingerprint to understand the plant physiological
responses to external fluctuations (Schurr et al., 2006). The
enzyme activity assay pipeline was employed in two case studies where the impact of external stimuli in vitro and in vivo has
been analysed. Thus, distinct enzymatic signatures could be
Carbohydrate enzyme activities for physiological phenotyping | 5533
established during inflorescence development in the monocot
Lolium multiflorum as well as in vitro cold treatment of young
inflorescences, a treatment related to doubled haploid formation. In addition, the impact of elevated CO2 concentrations,
which is an important parameter related to global climate
change, on enzymatic and hormone signatures for leaves of
the dicot Brassica napus was assessed, revealing a distinct
impact of CO2 concentration for both types of fingerprints.
Further, the enzyme activity assay pipeline was successfully
used to detect disctinct differences in various tissues and cell
types from two additional monocot and four additional dicot
model and crop species, exemplifying the broad applicability
and robustness of this method.
Materials and methods
Plant material and growth conditions
Sugar beet (Beta vulgaris L.), winter oilseed rape variety WüR03
(Brassica napus L.), Italian ryegrass cultivars LMW07/220 and
Andrea (Lolium multiflorum L.), barley cultivar Golden Promise
(Hordeum vulgare L.), tobacco SR1 (Nicotiana tabacum L.), and
tomato (Solanum lycopersicum L.) were grown in pots filled with
soil (Naturahum; Gramoflor, Vechten, Germany) under greenhouse
conditions at 20–24 °C achieved by additional heating during colder
outside temperatures, and a 16 h/8 h day/night cycle by supplementary illumination (Plug and Grow™ 200 W 6400 K fluorescent
lamps; Trade Hydro, Cleckheaton, UK). Perennial ryegrass cultivar Aberavon (Lolium perenne L.) was grown in vermiculite (AgraVermiculite; Pull Rhenen, Rhenen, The Netherlands) in growth
chambers (Heraeus; Vötsch, Balingen, Germany) at 20 °C/18 °C
(day/night) and a 16 h/8 h day/night cycle. Harvested plant material was frozen in liquid nitrogen and stored at –80 °C until further
use. The spring oilseed rape cultivar Campino (B. napus L.) was
grown in the field in a FACE (free-air CO2 enrichment) system (Erbs
and Fangmeier, 2006) under ambient (380 μl l–1) and elevated CO2
concentrations (613 μl l–1) (Oehme et al., 2013), and leaf material
(youngest elongated leaf) was harvested from five plants each. For
the analysis of inflorescence developmental stages from the Italian
ryegrass cultivars LMW07/220 and Andrea, inflorescences were harvested from at least 10 individual plants at the young inflorescence
(yi) stage with microspores in the late uninucleate stage, mature
inflorescence (mi) stage with binucleate microspores and all spikelets
emerged, and the old inflorescence (oi) stage with the anthers starting to open. The cold treatment of complete spikelets, isolated at the
yi stage from these cultivars was performed at 4 °C for 1–3 weeks.
Cell suspension cultures
Photoautotrophic Arabidopsis thaliana L. (Ap) (Hampp et al., 2012)
and tomato (S. lycopersicum L.) cells (Tp) (Roitsch and Sinha, 2002)
were cultivated in liquid medium consisting of 4.4 g l–1 MS0222
(Duchefa, Haarlem, the Netherlands) and 20 μg l–1 2,4-D (pH 5.7).
Heterotrophic tobacco (N. tabacum L.) Bright Yellow-2 (BY2)
cells were cultivated as described previously (Hyun et al., 2011).
Heterotrophic tomato cells (Th) were cultivated in C3-medium consisting of 4.4 g l–1 MS0222, 30 g l–1 sucrose, 0.17 g l–1 KH2PO4, 0.1 mg
l–1 2,4-D, and 20 μg l–1 kinetin (pH 6.0). Mixotrophic A. thaliana
cells (Am) were cultivated in medium consisting of 3.2 g l–1 MS0210,
20 g l–1 sucrose, and 1 mg l–1 2,4-D (pH 5.7). Cell suspension cultures
were grown at 26 °C, shaking at 125 rpm. Photoautotrophic and
mixotrophic cultures were grown under permanent light, and heterotrophic cultures under ambient light conditions. Enzyme activities
were analysed for 2-week-old Ap cultures, 10-day-old Tp cultures,
and 5-day-old BY2, Th, and Am cultures. Cells were pelleted by
10 min centrifugation at 10 000 g and 4 °C. Culture supernatants
were removed, and the cell pellet was frozen in liquid nitrogen and
stored at –80 °C until further use.
Chemicals
Substrates and auxiliary substances for enzyme activity
assays 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid)
(ABTS), ATP, glucose, fructose, NADP, sucrose, and uridine5′-diphosphoric acid (UDP) were purchased from AppliChem
(Darmstadt, Germany). Glucose-6-phosphate (G6P), NAD, and
NADH were purchased from Carl Roth (Karlsruhe, Germany).
Adenosine-5′-diphosphoglucose (ADPGlc), fructose-6-phosphate
(F6P), fructose-1,6-bisphosphate (F1,6bisP), glucose-1-phosphate
(G1P), glucose-1,6-bisphosphate (G1,6bisP), 3-phosphoglyceric acid (3-PG), and pyrophosphate (PPi) were purchased from
Sigma-Aldrich (Munich, Germany). Uridine-5′-diposphoglucose
(UDPGlc) was purchased from Wako Chemicals (Osaka, Japan).
Auxiliary enzymes for enzyme activity assays Glucose oxidase from
Aspergillus niger (GOD; EC 1.1.3.4; Serva, Heidelberg, Germany)
and peroxidase from horseradish (POD; EC 1.11.1.7; AppliChem,
Darmstadt, Germany) for invertase activity assays were purchased
as lyophilized powders. For kinetic enzyme activity assays, auxiliary enzymes were purchased as suspensions in 3.2 M ammonium
sulphate when available, or purchased as lyophilized powders and
suspended in 3.2 M ammonium sulphate at pH 6.0 before use. Ald
from rabbit muscle (EC 4.1.2.13), glycerol-3-phosphate dehydrogenase from rabbit muscle (GPDH; EC 1.1.1.8), and triosephosphate
isomerase from rabbit muscle (TPI; EC 5.3.1.1; lyophilized powder)
were purchased from Sigma-Aldrich (Munich, Germany). PGI from
yeast (EC 5.3.1.9) and HXK from yeast (EC 2.7.1.1) were purchased
from Roche (Mannheim, Germany). PGM from rabbit muscle
(EC 5.4.2.2) was purchased from Megazyme (Wicklow, Ireland).
G6PDH from Leuconostoc mesenteroides (EC 1.1.1.49; lyophilized
powder) was purchased from Oriental Yeast (Tokyo, Japan).
Chemicals for extraction buffers and assay buffers TRIS, HEPES,
K2HPO4, KH2PO4, citric acid, Na2HPO4, β-mercaptoethanol,
MgCl2, HCl, NaOH, NADH, and G6P were purchased from Carl
Roth (Karlsruhe, Germany). Benzamidine, BisTris, bovine serum
albumin (BSA), dithiothreitol (DTT), EDTA, glycerol, NaCl, and
phenylmethylsulphonyl fluoride (PMSF) were purchased from
AppliChem (Darmstadt, Germany).
Other chemicals Polyvinylpolypyrrolidone (PVPP) was purchased from Sigma-Aldrich (Munich, Germany). Roti®-Quant 5×
concentrate Bradford reagent for determination of protein content (Bradford, 1976) was purchased from Carl Roth (Karlsruhe,
Germany).
Extraction procedure
Frozen plant material was homogenized in liquid nitrogen with
0.1% PVPP (the required amount may vary depending on the type
of plant material). Routinely, 500 mg of plant material was used for
extractions and all subsequent steps were performed on ice and/or
in cooled incubation rooms (4 °C), using pre-cooled liquids. For
comparison of extraction buffers, the ground plant material was
extracted with either 1 ml of extraction buffer A (200 mM HEPES/
NaOH pH 7.5, 3 mM MgCl2, 1 mM EDTA, 2% glycerol, 0.1 mM
PMSF, 1 mM benzamidine; Bonfig et al., 2010) or 1 ml of extraction
buffer B (40 mM TRIS-HCl pH 7.6, 3 mM MgCl2, 1 mM EDTA,
0.1 mM PMSF, 1 mM benzamidine, 14 mM β-mercaptoethanol,
24 μM NADP; Bisswanger, 2004) on ice until material was fully
thawed (20–60 min, depending on the type of plant material; see
Fig. 1). The homogenate was centrifuged at 4 °C and 20 000 g for
5–30 min until a solid pellet was obtained (depending on the type
of plant material), and the pellet was kept on ice. The supernatant
(crude extract; soluble proteins) was subsequently centrifuged for up
to 45 min (depending on the type of plant material) at 4 °C and 20
000 g to remove all remaining particles. The pellet was washed three
times with distilled water, re-suspended in either 1 ml of high salt
5534 | Jammer et al.
buffer A (1 M NaCl, 200mM HEPES/NaOH pH 7.5, 3 mM MgCl2,
1 mM EDTA, 2% glycerol, 0.1 mM PMSF, 1 mM benzamidine;
Bonfig et al., 2010) or 1 ml of high salt buffer B (1 M NaCl, 40 mM
TRIS-HCl pH 7.6, 3 mM MgCl2, 1 mM EDTA, 0.1 mM PMSF,
1 mM benzamidine, 14 mM β-mercaptoethanol, 24 μM NADP;
modified from Bisswanger, 2004), and mixed by continuous shaking
at 4 °C overnight. The homogenate was centrifuged at 4 °C and 20
000 g for 5–30 min (depending on the type of plant material). The
supernatant (cell wall extract; proteins that were ionically bound
to the cell walls) was subsequently centrifuged for up to 45 min
(depending on the type of plant material) at 4 °C and 20 000 g to
remove all particles. The cell wall extract and a fraction of the crude
extract were dialysed overnight against 20 mM potassium phosphate
buffer (pH 7.4) at 4 °C (for an overview of the extraction procedure, see Fig. 1). The Susy, HXK, FK, and invertase enzymes use
substrates that are highly abundant in non-dialysed protein extracts.
Therefore, extracts that are not dialysed lead to high background, in
both sample and blank preparations. Further, dialysis is required for
invertases, since the β-mercaptoethanol used in the extraction buffer
inhibits POD activity in the second part of the assay procedure.
Optionally, extract protein contents were determined according
to the Bradford method (Bradford, 1976), using BSA as a standard
protein. All extracts were snap-frozen in liquid nitrogen and stored
at –20 °C in small aliquots until further use. The 13 selected enzyme
activity assays were performed according to one of the four basic
reaction schemes (Fig. 2), and represent the key enzymes of primary
carbohydrate metabolism (Fig. 3). An overview of the respective
assay components is provided in Supplementary Table S1 available
at JXB online.
Invertase activity assay
Invertase activity was assayed in a miniaturized end-point assay
based on the method of Sung et al. (1989). Aliquots (up to 20 μl)
of dialysed crude extract or cell wall extract were incubated at
37 °C for 30 min in flat bottom 96-well plates (Sarstedt, Nümbrecht,
Fig. 1. Flowchart of the universal protein extraction and dialysis protocol.
All steps are performed on ice and/or in cooled incubation rooms (4 °C).
1
The required amount of PVPP may vary depending on the type of plant
material. 220–60 min incubation on ice, depending on the type of plant
material. 3Centrifugation at 4 °C for 5–30 min until a solid pellet is obtained,
depending on the type of plant material. 4Centrifugation at 4 °C for up to
45 min until clear supernatant is obtained, depending on the type of plant
material.
Germany) with 5 μl of 100 mM sucrose and 5 μl of reaction buffer
pH 4.5 (454 mM Na2HPO4/273 mM citric acid) or pH 6.8 (772 mM
Na2HPO4/114 mM citric acid) for vacuolar invertase (vacInv) and
cell wall invertase (cwInv) or cytoplasmic invertase (cytInv), respectively, adding distilled water to a total reaction volume of 50 μl.
Glucose standards (0–50 nmol) were treated accordingly for the
respective calibration curves. All measurements were carried out in
triplicate. For control reactions, sucrose was omitted. The amount
of liberated glucose was determined by measurement of absorbance at 405 nm in a plate reader (Ascent Multiskan; Thermo Fisher
Scientific, Waltham, MA, USA) after 30 min of incubation at room
temperature with 200 μl of GOD-POD reagent (10 U ml–1 GOD,
0.8 U ml–1 POD, and 0.8 mg ml–1 ABTS in 0.1 M potassium phosphate buffer, pH 7.0). Specific activities were expressed as nkat mg
protein–1 or nkat g FW–1.
Kinetic enzyme activity assays
General protocol All kinetic enzyme activity assays were established in UV-transmissive single-use microcuvettes (Plastibrand®;
Brand, Wertheim, Germany) in a total reaction volume of 200 μl in
a spectrophotometer (U-3000; Hitachi, Tokyo, Japan). Assays were
subsequently adapted to a 96-well microtitre plate format for semihigh-throughput application. For routine measurement, aliquots
(up to 25 μl) of the different protein extracts were incubated in a
plate reader (Ascent Multiskan; Thermo Fisher Scientific) at 30 °C
for 20 min in UV-transmissive flat bottom 96-well plates (UV-Star;
Greiner Bio One, Kremsmünster, Austria) in a total reaction volume
of 160 μl with a mixture of buffer components, substrate(s), auxiliary substance(s), and auxiliary enzymes (Fig. 2; Supplementary
Table S1 at JXB online), and absorbance at 340 nm was monitored
throughout the entire period of incubation. All assays were carried
out in triplicate. For control reactions, substrate was omitted. The
change in absorbance per second during the linear phase of substrate conversion was used as the basis for the calculation of specific
enzyme activity in nkat mg protein–1 or in nkat g FW–1.
Specific protocols for individual enzymes (in alphabetical order) For
determination of AGPase activity, aliquots of untreated crude
extract were incubated with 0.44 mM EDTA, 5 mM MgCl2, 0.1%
BSA, 2 mM ADPGlc, 1.5 mM PPi, 1 mM NADP, 2 mM 3-PG,
0.432 U of PGM, and 1.28 U of G6PDH in 100 mM TRIS-HCl at
pH 8.0 [modified from Pelleschi et al. (1997) and Appeldoorn et al.
(1999)]. For control reactions, ADPGlc was omitted. The increase
in absorbance at 340 nm due to conversion of NADP to NADPH
was monitored.
For determination of Ald activity, aliquots of untreated crude
extract were incubated with 1 mM EDTA, 5 mM MgCl2, 1 mM
F1,6bisP, 0.15 mM NADH, 0.48 U of TPI, and 0.8 U of GPDH in
50 mM TRIS-HCl at pH 8.0 (modified from Schwab et al., 2001). For
control reactions, F1,6bisP was omitted. The decrease in absorbance
at 340 nm due to conversion of NADH to NAD was monitored.
For determination of FK activity, aliquots of dialysed crude
extract were incubated with 5 mM MgCl2, 5 mM fructose, 2.5 mM
ATP, 1 mM NAD, 0.8 U of PGI, and 0.8 U of G6PDH in 50 mM
BisTris at pH 8.0 [modified from Appeldoorn et al. (1999) and
Petreikov et al. (2001)]. For control reactions, fructose was omitted.
The increase in absorbance at 340 nm due to conversion of NAD to
NADH was monitored.
For determination of G6PDH activity, aliquots of untreated
crude extract were incubated with 5 mM MgCl2, 1 mM G6P, and
0.4 mM NADP in 100 mM TRIS-HCl at pH 7.6 (modified from
Bisswanger, 2004). For control reactions, G6P was omitted. The
increase in absorbance at 340 nm due to conversion of NADP to
NADPH was monitored.
For determination of HXK activity, aliquots of dialysed crude
extract were incubated with 5 mM MgCl2, 5 mM glucose, 2.5 mM
ATP, 1 mM NAD, and 0.8 U of G6PDH in 50 mM BisTris at pH
8.0 [modified from Appeldoorn et al. (1999) and Petreikov et al.
(2001)]. For control reactions, glucose was omitted. The increase in
Carbohydrate enzyme activities for physiological phenotyping | 5535
Fig. 2. Reaction schemes to determine the 13 selected enzyme activities. (A) Invertase enzymes. The oxidized ABTS is detected by its green colour. (B)
UGPAse and AGPase. (C) FK, HXK. PGI, PGM, G6PDH, and Susy. (D) PFK and Ald.
Fig. 3. Key enzymes of primary carbohydrate metabolism. The 13 selected enzymes (underlined) are key enzymes of primary carbohydrate metabolism,
among which cell wall invertase (cwInv), vacuolar invertase (vacInv), cytoplasmic invertase (cytInv), and sucrose synthase (Susy) possess sucrolytic
activity, fructokinase (FK) is important for sucrose biosynthesis; hexokinase (HXK), phosphoglucoisomerase (PGI), phosphofructokinase (PFK), and
aldolase (Ald) are important for glycolysis; ADP-glucose pyrophosphorylase (AGPase) and phosphoglucomutase (PGM) are important for starch
biosynthesis; UDP-glucose pyrophosphorylase (UGPase) is important for cell wall biosynthesis; and glucose-6-phosphate dehydrogenase (G6PDH) is
important for the oxidative pentose phosphate pathway. The figure is based on the design in Sergeeva and Vreugdenhil (2002).
absorbance at 340 nm due to conversion of NAD to NADH was
monitored.
For determination of PFK activity, aliquots of untreated crude
extract were incubated with 1 mM EDTA, 5 mM MgCl2, 1 mM F6P,
0.2 mM ATP, 0.15 mM NADH, 0.16 U of aldolase, 0.48 U of TPI,
and 0.8 U of GPDH in 50 mM TRIS-HCl at pH 8.0 (modified from
Klotz et al., 2006). For control reactions, F6P was omitted. The
decrease in absorbance at 340 nm due to conversion of NADH to
NAD was monitored.
For determination of PGI activity, aliquots of untreated crude
extract were incubated with 4 mM MgCl2 (exception: 0.1 mM
for sugar beet PGI), 4 mM DTT, 2 mM F6P, 0.25 mM NAD, and
0.32 mM G6PDH in 100 mM TRIS-HCl at pH 8.0 (modified from
Zhou and Cheng, 2008). For control reactions, F6P was omitted.
The increase in absorbance at 340 nm due to conversion of NAD to
NADH was monitored.
For determination of PGM activity, aliquots of untreated crude
extract were incubated with 10 mM MgCl2 (exception: 0.1 mM for sugar
5536 | Jammer et al.
beet PGM), 4 mM DTT, 0.1 mM G1,6bisP, 1 mM G1P (exception:
3 mM for sugar beet PGM), 0.25 mM NAD, and 0.64 U of G6PDH in
20 mM TRIS-HCl at pH 8.0 (modified from Manjunath et al., 1998).
For control reactions, G1P was omitted. The increase in absorbance at
340 nm due to conversion of NAD to NADH was monitored.
For determination of Susy activity, two reactions were performed,
(A) including 1 mM UDP detecting both Susy and cytInv background activity, and (B) without 1 mM UDP to detect the cytInv
background activity only. Susy activity was calculated by subtracting cytInv background activity (B) from total activity (A). For both
reactions, aliquots of dialysed crude extract were incubated with
1 mM EDTA, 2 mM MgCl2, 5 mM DTT, 250 mM sucrose, 1 mM
UDP (omitted for reaction B), 1.3 mM ATP, 0.5 mM NAD, 0.672 U
of HXK, 0.56 U of PGI, and 0.32 U of G6PDH in 50 mM HEPES/
NaOH at pH 7.0 (modified from Pelleschi et al., 1997). For control reactions, sucrose was omitted. The increase in absorbance at
340 nm due to conversion of NAD to NADH was monitored.
For determination of UGPase activity, aliquots of untreated crude
extract were incubated with 0.44 mM EDTA, 5 mM MgCl2, 0.1% BSA,
2 mM UDPGlc, 1.5 mM PPi, 1 mM NADP, 2 mM 3-PG, 0.432 U of
PGM, and 1.28 U of G6PDH in 100 mM TRIS-HCl at pH 8.0 [modified from Pelleschi et al. (1997) and Appeldoorn et al. (1999)]. For control reactions, UDPGlc was omitted. The increase in absorbance at
340 nm due to conversion of NADP to NADPH was monitored.
Miniaturization and adaptation of the activity assays to a 96-well
multi-well format
Following establishment of the different enzyme assays, the protocols, typically designed for a relatively large volume of 3 ml, were
miniaturized and adapted to a 96-well microtitre format, in order
to reduce costs for chemicals, the amount of plant material required
and, most importantly, hands-on time. Routinely, each sample is
measured as three technical replicates on one plate using the same
reaction mix to ensure robustness and reproducibility, while substrates were omitted in negative controls. Since one person is able to
process five 96-well plates per day with 20 samples each, equivalent
to 100 samples per day, but only five samples using single cuvets
(three technical replicates and one blank), at least 20-fold more
sample measurements can be processed per person in 96-well plates.
Typically, between 2.5 μl and 25 μl of extract was sufficient to determine enzymatic activities within the linear range, which was reached
after a variable lag phase and in some cases reached saturation
towards the end of a measurement. Therefore, each activity graph
was manually inspected to specify the linear phase from which the
kinetic data for the calculation of enzyme activity were obtained.
Following protein extraction and optional dialysis, the extracts were
split into small aliquots and snap-frozen in liquid nitrogen for storage at –20 °C. These samples were only thawed once, which proved
to deliver optimal, robust, and reproducible data.
Hormone analysis
For the analysis of phytohormone levels in the leaves of spring oilseed rape cultivar Campino, extractions were performed using 100 mg
of frozen and ground leaf tissue, after adding 4 μl of internal standard mix composed of deuterium-labelled hormones. Phytohormone
detection was performed on a UHPLC–MS/MS system consisting of
a Thermo ACCELA pump (Thermo Scientific) coupled to a tempered
HTC-PAL autosampler (CTC Analytics, Zwingen, Switzerland),
and connected to a Thermo TSQ Quantum Access Max Mass
Spectrometer (Thermo Scientific) with a heated electrospray ionization (HESI) interface, using a. Nucleoshell-PFP column (2.7 μm,
100 × 2 mm; Macherey-Nagel) according to Großkinsky et al. (2014).
Statistical analysis
Standard deviations and average values were calculated in Excel.
Statistical significance for differences between treatments was analysed using the Student’s t-test in SPSS at the P<0.05 level.
Results and Discussion
To establish an analytical platform for the characterization of the physiological state of model and crop plants, 13
key enzymes of primary carbohydrate metabolism (Fig. 3;
Supplementary Table S1 at JXB online) were selected for the
determination of an activity signature profile. The selected
enzymes are involved in the metabolism of the transport
sugar sucrose, ranging from cleavage into hexose monomers
and metabolism via glycolysis and the oxidative pentose
phosphate pathway to the (re-)synthesis of starch.
Comparison of buffers for the preparation of crude and
fractionated protein extracts
The routine and systematic screening for differential changes
in enzymatic activities requires simple and fast methods.
Therefore, initially the applicability of one uniform extraction
method was tested for the selected enzymes, comparing two
buffers. Buffer A is a standard buffer for the extraction and
subsequent analysis of invertase enzyme activities (Bonfig
et al., 2010), while buffer B was described to be essential for
G6PDH activity (Bisswanger, 2004). For the majority of the
analysed plant species and organs, several enzymes showed
significantly higher activities using buffer B (Fig. 4), which
was therefore subsequently used as the standard extraction
buffer.
Differential requirement for dialysis of the crude
extracts for different enzyme assays
Next, it was analysed whether residual components in the
crude extracts influenced enzyme activity assays (Kruger,
1995). The crude protein extracts were dialysed against phosphate buffer to remove contaminations such as ions and sugars. The comparison of crude versus dialysed extracts showed
that the invertase, Susy, HXK, and FK enzymes required
dialysis prior to the assays (Supplementary Table S1 at JXB
online). Therefore, the preparation of the protein extracts
Fig. 4. Comparison of protein extraction buffers. Enzyme activities
were compared between protein extracts prepared with extraction
buffer A (black) or B (white), respectively, from the same plant material
(mature leaf of L. perenne). Specific activities in nkat gFW–1 are given as
mean values from n=3 measurements of the same plant extract ±SD.
Preparations were extracted in either buffer, each for single biological
samples consisting of at least five individual plants. Significant differences
in activities between extraction with buffer A or B are indicated (*P<0.05;
**P<0.01).
Carbohydrate enzyme activities for physiological phenotyping | 5537
for all assays can be performed with one extraction buffer,
and, depending on the enzyme, this crude extract can either
be directly employed for activity assays or dialysed prior to
analysis, while the pellet left after the crude extraction is used
to isolate the cell wall-bound fraction (cwInv).
Adoption and optimization of enzyme activity assays
The selected enzyme assays were established as single cuvette
assays based on the original published procedures (Pelleschi
et al., 1997; Manjunath et al., 1998; Appeldoorn et al.,
1999; Petreikov et al., 2001; Schwab et al., 2001; Bisswanger,
2004; Klotz et al., 2006; Zhou and Cheng, 2008). To avoid
the pitfalls discussed for coupled spectrophotometric assays
(Kruger, 1995), the different assays were established employing extensive controls, including incubation time, substrate
concentration, and amount of extract in different plant species to assess the optimal assay conditions within the linear
phase of activity in the assays. Both technical and biological
replicates were assayed, to assess the robustness and reproducibility of the extraction and assay protocols. Separate
measurements for repeated extractions from the same plant
material, as well as repeated measurements for the same sample were performed to obtain additional technical replicates.
Serial experiments carried out under controlled conditions
were performed to obtain biological replicates. For invertase
activities, pH optima were additionally determined. Cell wall
invertase assay conditions in L. perenne are shown as exemplary assay optimization (Fig. 5). In cases where the obtained
enzyme activities were obviously much lower than comparable organs/developmental stages in other plant species,
and therefore potentially underestimated, additional assay
parameters were assessed for optimization. Thus, it was identified that the standard assay conditions for PGM and PGI
require a specific optimization in B. vulgaris. Whereas the
MgCl2 concentration needs to be adjusted for both assays
[0.1 mM instead of 10 mM (PGM) or 4 mM (PGI)], the G1P
concentration need to be adjusted only for the PGM assay
(3 mM instead of 1 mM). For L. perenne, the pH optima for
vacInv and cwInv differed slightly from the standard (pH 4.7
instead of 4.5).
Typically, the enzyme activities were expressed relative to
fresh weight (nkat g FW–1) and activities were only expressed
relative to protein amount (nkat mg protein–1) when the protein concentrations are likely to vary considerably between
the samples that were compared, such as direct comparisons
between different organs or different developmental stages.
Case study 1: cold treatment of ryegrass
inflorescences used for doubled haploid generation
results in distinct changes in carbohydrate enzyme
activity signatures
To evaluate the potential of the established experimental
platform for the determination of a signature of key enzymes
involved in carbohydrate metabolism, two case studies were
performed. In these studies, it was analysed whether developmental stages and the impact of external stimuli are reflected
by a differential enzyme activity signature.
The first set of expriments involved the determination of
the enzyme activities in inflorescences of L. multiflorum. The
process of doubled haploid formation is of high importance
Fig. 5. Optimization of cell wall invertase assay conditions. Enzyme activities for cell wall invertase in extracts made from L. perenne mature leaf (ML)
and stubble (ST), determined under different assay conditions. Specific activities in nkat gFW–1 are given as mean values from n=3 measurements of the
same plant extract ±SD. Preparations of each plant extract consisted of at least five individual plants. (A) Analysis of the pH optimum in two independent
biological replicates of L. perenne (ML) showing optimum pH at 4.7. (B) Analysis of time dependence of L. perenne (ML) showing linearity for 30 min and
45 min. (C) Analysis of substrate linearity of L. perenne (ML) showing linearity between 0 and 40 mM sucrose. (D) Analysis of linearity for the amount of
extract in two independent biological replicates of L. perenne (ST) showing linearity between 2.5 μl and 10 μl of extract.
5538 | Jammer et al.
to accelerate plant breeding and is based on the regeneration
of diploid homozygous plants from haploid gametes, often
from reprogrammed microspores. Typically the empirically
developed in vitro protocols include a nutrient depletion, indicating a critical role for carbohydrate metabolism. In addition, the developmental stage of the microspores seems to be
critical, and species-specific differences have been observed
(Islam and Tuteja, 2012). The carbohydrate enzyme activity
signatures were studied in two cultivars of L. multiflorum during inflorescence development as well as in vitro cold treatment of young inflorescences, a treatment shown to promote
doubled haploid formation (Grewal et al., 2009).
The analysis of carbohydrate enzyme activity signatures
during three inflorescence developmental stages (Table 1;
Supplementary Fig. S1 at JXB online) revealed considerable differences between the cultivars LMW07/220 and
Andrea. The majority of the enzymes showed higher activities for cultivar Andrea during inflorescence development,
whereas only HXK and FK activities were higher for cultivar LMW07/220 at the youngest inflorescence stage. In contrast, invertase activities were similar for both cultivars. Cold
treatment of young inflorescences reduced the differences in
HXK, FK, and G6PDH activities between the two cultivars,
while the higher activities for PGI, PGM, UGPase, Ald, and
PFK in Andrea were maintained (Table 1; Supplementary
Fig. S2). Cold treatment in general reduced invertase activities in both cultivars, which could be attributed to (nutrient)
stress-induced doubled haploid formation. The process of
doubled haploid formation is still poorly understood and the
protocols for the treatment of tissues used are determined
empirically. Thus, the determination of a comparative physiological fingerprint of contrasting genotypes with differential potential for doubled haploid generation will provide a
basis for a more systematic approach to compare the enzyme
activity signatures with the yield in doubled haploids. This
can lead to the identification of a pre-disposition for the
potential for doubled haploid generation and be used as a
physiological marker to optimize the treatments, select the
most suitable cultivars, and establish protocols for recalcitrant species.
Case study 2: elevated CO2 concentrations have a
differential impact on the enzyme activity signature in
oilseed rape leaves
Atmospheric CO2 concentration is predicted to reach 550 μl l–1
in the middle of this century (IPCC, 2013). This increase has
been shown to affect plant physiology, morphology, development, growth, and reproduction (Bazzaz, 1990). Spring oilseed
rape grown under elevated CO2 concentration using a FACE
facility showed an increase in biomass, dry weight, stem and
shoot length, and leaf area (Franzaring et al., 2008; Högy
et al., 2009), as well as changes in allocation patterns, C:N,
ratios and source–sink relationships (Franzaring et al., 2011,
2012). To address whether an impact of elevated CO2 concentration is also evident at the level of a physiological trait, the
activity signature of the key carbohydrate-related enzymes was
determined.
The elevated CO2 concentration and thus changed C:N
ratio is reflected at the enzyme activity signature and had
a differential impact on the different enzyme activities.
Sucrolytic enzyme activities were reduced (Fig. 6A–D),
most prominently for cwInv and cytInv (P=0.063). G6PDH
activity was not significantly affected (Fig. 6E). Among the
enzymes involved in glycolysis, HXK activity was unaffected
(Fig. 6F), both Ald (Fig. 6G) and PFK (Fig. 6H) activities
were reduced, while PGI activity was increased (Fig. 6I), albeit
not significantly. The AGPase (Fig. 6J) and PGM (Fig. 6K)
activities involved in starch biosynthesis were weakly but not
significantly increased. Interestingly, leaf starch levels (data
not shown) were 53.1% higher under elevated CO2 concentration. Among the enzymes involved in sucrose biosynthesis,
FK activity was unaffected (Fig. 6L), while UGPase activity
was slightly increased (Fig. 6M, P=0.073).
Table 1. The impact of inflorescence developmental stage and cold treatment of immature inflorescences on enzyme activity signatures
in two cultivars of Lolium multiflorum
Pathway
Sucrolytic
Glycolysis
Starch
Sucrose biosynthesis
Oxidative pentose phosphate
Enzyme
cwInv
vacInv
cytInv
Susy
HXK
Ald
PFK
PGI
PGM
FK
G6PDH
Development
Cold treatment
LMW07/220
Andrea
LMW07/220
Andrea
Up
Up
Up
Down
Down
Down
Down
Down
No change
Down
No change
Up
Up
Up
No change
Down
No change
Transient up
No change
No change
No change
Transient up
Down
Down
Down
Down
Transient up
Transient up
Transient up
No change
Down
Transient up
No change
Down
Down
Down
No change
Up
Up
Up
No change
Transient up
Transient up
Down
Abbreviations: AGPase, ADP-glucose pyrophosphorylase; Ald, (fructose 1,6-bisphosphate) aldolase; cwInv, cell wall invertase;
cytInv, cytoplasmic (neutral) invertase; FK, fructokinase; G6PDH, glucose-6-phosphate dehydrogenase; HXK, hexokinase; PGI,
phosphoglucoisomerase; PGM, phosphoglucomutase, PFK, phosphofructokinase all enzymes; SD, standard deviation; Susy, sucrose synthase;
UGPase, UDP-glucose pyrophosphorylase; vacInv, vacuolar invertase.
Carbohydrate enzyme activities for physiological phenotyping | 5539
Fig. 6. The impact of elevated CO2 concentration on enzyme activities in leaves from Brassica napus. Leaf enzyme activities in spring oilseed rape plants
cultivar Campino, grown under ambient (380 μl l–1) or elevated (613 μl l–1) CO2 concentration for (A) cytInv, (B) vacInv, (C) cwInv, (D) Susy, (E) G6PDH, (F)
HXK, (G) Ald, (H) PFK, (I) PGI, (J) AGPase, (K) PGM, (L) FK, and (M) UGPase. Specific activities in nkat gFW–1 are given as mean values ±SD from five
biological replicates measured as three technical replicates each. Significant differences between CO2 concentrations are indicated (*P<0.05; **P<0.01).
To demonstrate the implementation of an enzyme activity signature within a holistic physiological phenotyping
approach, phytohormone profiles were also determined
from the same set of samples. Plant hormones are important not only for plant growth, but also for responses to
environmental cues, and are involved in the regulation of
metabolism, especially invertase enzyme activities. Recently,
the importance of analysing hormone signatures for the
physiological characterization of plant responses was shown
(Großkinsky et al., 2014). Therefore, the impact of elevated
CO2 concentration on the hormonal fingerprint of oilseed
rape leaves was also investigated. In general, cytokinin
levels were reduced (Fig. 7A–F), most evidently for transzeatin-O-glucoside (Fig. 7A), trans-zeatin riboside (Fig. 7C,
P=0,089), and total cytokinin levels (Fig. 7F). These lower
cytokinin levels could have caused the reduced sucrolytic
enzyme activities under elevated CO2 concentration, since
invertases were shown to be regulated by cytokinins (Ehneß
and Roitsch, 1997; Balibrea Lara et al., 2004). Among the
acidic hormones (Fig. 7G–J), only abscisic acid levels were
reduced (Fig. 7I, P=0,066), which could reflect delayed leaf
senescence under elevated CO2 concentration. These data
indicate that the comparative determination of the enzyme
activity as well as the phytohormone signatures is valuable
for the systematic analysis of a physiological fingerprint and
to decipher the underlying regulatory mechanisms.
Application of the method for the determination of
carbohydrate enzyme activity signatures to various
monocotyledonous and dicotyledonous model and
crop plants and tissues
In a further set of experiments, it was investigated whether
the developed pipeline for determination of an enzyme activity signature can be generally applied for routine physiological phenotyping of a variety of species without the need for
specific adaptations and optimizations. Thus, the carbohydrate enzyme signatures was determined in vegetative, reproductive and storage organs at different developmental stages
from additional monocot (H. vulgare and L. perenne) and
dicot (B. vulgaris, B. napus, S. lycopersicum, and N. tabacum)
plant species, including several crop species (Supplementary
Tables S2, S3 at JXB online). Since plant cell cultures represent a valuable experimental system, these assays were
also employed to analyse signatures in heterotrophic, mixotrophic, and autotrophic cell cultures from S. lycopersicum,
N. tabacum, and A. thaliana (Supplementary Table S4). Most
enzymes could reliably be detected in all plant species, organs,
and cell suspensions analysed. In general, the highest enzyme
activities and largest differences were obtained for the three
invertases, as well as PGM and PGI. In contrast, AGPase and
Susy predominantly showed very low activities. Due to the
high substrate costs, AGPase assays were only performed for
selected plant species/organs. Susy only showed high levels of
5540 | Jammer et al.
Fig. 7. The impact of elevated CO2 concentration on hormone levels in leaves from Brassica napus. Leaf hormone levels in spring oilseed rape plants,
cultivar Campino, grown under ambient (380 μl l–1) or elevated (613 μl l–1) CO2 concentration for (A) trans-zeatin-O-glucoside, (B) trans-zeatin ribosideO-glucoside, (C) trans-zeatin riboside, (D) dihydrozeatin riboside, (E) isopentenyladenine, (F) total cytokinins, (G) indole-3-acetic acid, (H) salicylic acid,
(I) abscisic acid, and (J) jasmonic acid. Hormone levels in ng gFW–1 are given as mean values ±SD from five biological replicates measured as three
technical replicates each. Significant differences between CO2 concentrations are indicated (*P<0.05).
activity in older B. vulgaris tap roots, whereas activities for the
three invertases are comparatively low, which is in agreement
with earlier studies (Godt and Roitsch, 2006).
Conclusions and perspectives
In this study, a uniform method suitable for the preparation
of crude protein extracts from a large variety of plant species
and organs is presented. These extracts were used to establish
an experimental platform for the quantitative measurement
of enzyme activities for 13 key enzymes of primary carbohydrate metabolism using coupled spectrophotometric assays
in a 96-well format, enabling semi-high-throughput analysis.
With only a few exceptions, the enzyme assay conditions are
uniform and could be applied to each of the range of plant
species and organs analysed, which offers several advantages
over the original protocols. First, a limited amount of plant
material (routinely 500 mg) is needed since only one extraction is performed. Secondly, the costs are reduced since
smaller assay volumes are required. Thirdly, labour time is
reduced since ~20-fold more assays per day can be performed
in this semi-high-throughput format to analyse one particular
enzyme in 100 samples per day. Although the manual control
over the specification of the linear enzyme activity phase is
somewhat more time-consuming compared with end-point
measurements such as employed in the published robotics
platform (Gibon et al., 2004; Sulpice et al., 2010), this allows
an optimal and more precise determination of enzyme activities. It is possible to check the quality of any reaction with
respect to linearity of the kinetic assays, thus providing the
possibility to identify eventual negative factors directly when
analysing novel species. Fourthly, the specificity for changes
in enzymatic activities between samples can be determined
using the other enzymes as (normalization) controls. This
minimizes artefacts resulting from the extraction and assay
procedures, which are difficult to detect when different optimized extraction procedures are used for each individual
enzyme activity. The crude fractionation in an intracellular and a cell wall fraction allows conclusions on subcellular compartmentation. The determination of three types
of invertase activities shows the potential to discriminate
between isoenzymes based on the subcellular localization
and particular properties such as a differential pH optimum.
Also a differential substrate or product regulation or differential inhibitor sensitivity could be used to discriminate
between isoenzymes. Last but not least, the described system works independently of a robot-based procedure, which
would require a high investment in the necessary non-standard equipment, whereas typically plate readers are present in
biochemical–physiological laboratories. Thus, an affordable
enzyme activity phenotyping protocol has been established,
which is relevant for understanding plant performance at a
physiological level in addition to non-invasive phenotyping
techniques.
The enzyme activity signatures have been been applied in
two case studies to analyse the impact of external stimuli
applied in vivo in the field and in vitro. Distinct and differential changes in the enzyme activity signatures were apparent during Italian ryegrass inflorescence development and in
response to in vitro cold treatment of the reproductive tissues as a basis for doubled haploid generation. Likewise, the
treatment of field-grown oilseed rape plants by elevated CO2
concentration, related to the expected rise within the next
35 years, also resulted in distinct and differential changes
of the determined physiological fingerprint in the leaves.
Thus, the physiological phenotyping by the determination of
enzyme activity signatures for the two case studies delivered
valuable new information on the physiological changes during these two processes studied and complements other analytical approaches. The sensitivity of this method is apparent
Carbohydrate enzyme activities for physiological phenotyping | 5541
in particular from the fact, that in the case of the study on
the impact of elevated CO2 administered by a FACE facility
in the field, a range of other measured parameters were not
affected, despite an observed biological impact on the abundance of some species of insects (Oehme et al., 2013).
In summary, the established carbohydrate enzyme activity
pipeline offers a low cost and low tech method, which could
be employed by any lab, while no or only small adjustments
in the assay conditions are required for plant species and/or
organs other than those addressed by the present analyses.
This semi-high-throughput approach is very well suited for
physiological phenotyping to assess the physiological state
of model and crop plant species in combination with other
physiological trait parameters, such as the determination of
phytohormone signatures (Großkinsky et al., 2014). Data
infrastructures and tools for the comprehensive meta-analyses of the various sets of experimental data will need to be
generated. This will narrow the genotype–phenotype knowledge gap relevant for both basic research and plant breeding
to assess and elucidate the basis for the phenotypic plasticity
in relation to the multifactorial biotic and abiotic environment and agricultural management practice.
Supplementary data
Supplementary data are available at JXB online.
Figure S1. Enzymatic signatures during development of
Lolium multiflorum inflorescence samples.
Figure S2. Enzymatic signatures following cold treatment
of young Lolium multiflorum inflorescence samples.
Table S1. Overview of assay conditions for the selected
enzymes in carbohydrate metabolism.
Table S2. Enzyme activities in different organs from several
plant species based on protein amount.
Table S3. Enzyme activities in different organs from several
plant species based on fresh weight.
Table S4. Enzyme activities in various cell suspension
cultures.
Acknowledgements
Expert technical assistance by Ursula Janschek, Anna Luidold, and Tanja
Mayer is gratefully acknowledged. This work was supported by the Deutsche
Forschungsgemeinschaft (SFB) to NLE, KWS Saat AG to AJ, BMWF to
AG, and GFP Bank to EH and HC. We thank Eva Rosenqvist and Fulai Liu
for critical reading of the manuscript.
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