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Carbon mobilization at early maize embryo germination Sánchez-Nieto Sa, Sánchez-Linares La, Gavilanes-Ruíz Ma, Díaz-Pontones Db, Guzmán-Chávez Fa, Calzada-Alejo Va, Zurita-Villegas Va, Luna-
Loaiza Va, Moreno-Sánchez Rc.
a Dpto. de Bioquímica, Facultad de Química, Conjunto E. Universidad Nacional, Autónoma de México, Cd. Universitaria, Coyoacán. México 04510, D.F., México. bDpto. de Ciencias de la Salud, División de Ciencias Biológicas y de la Salud,Universidad Autónoma Metropolitana Iztapalapa. Apartado Postal 55535, México
09340, D.F., México. cDpto. de Bioquímica, Instituto Nacional de Cardiología Ignacio Chávez. Tlalpan, México 14080, D.F., México. E-mail: [email protected]
Maize embryos
Metabolite extraction
and analysis
1. Carbohydrates
2. Tryglicerides
Enzyme obtention and
activity determination
1. Hexokinase
II. Cell wall invertase
III. PM H+-ATPase
Localization of molecules
I. Sucrose histochemistry
II. Immunohistochemistry of transporters
Uptake detection
1. Sucrose and hexose(quantified by liquid
scintillation)
2. Oxygen
MATERIAL AND METHODS
INTRODUCTION
RESULTS
The maize embryo germination is not
affected by removing the endosperm (Fig
2A), and follows the typical triphasic curve of
water and oxygen uptake, with the two
phases within the 0 and 24 h of imbibition
(Fig 2 B). The radicle growth was maximal
about the 18 h (Fig 2C). Then for the
subsequent experiments the embryos were
imbibed for 0, 6, 12, and 24 h.
Figure 3. Triglyceride content and the invertase
and hexokinase activities during maize embryo
germination. (A) Sucrose localization in embryos
imbibed at different times. Arrows indicate sucrose-
stained regions. (B) Cell wall-invertase and
hexokinase activities. (C) Triglyceride content.
Figure 4. Sucrose (A) and glucose uptake (B) by
embryo or embryo axes during imbibition (A).
Figure 5. Immunolocalization of the sucrose transporter in maize embryos. Embryos were imbibed for 24
h, fixed, infiltrated in sucrose and frozen. Then, tissues were sliced into 8 m sections and slices were
processed for immunolocalization with the sucrose transporter antibody and viewed in a Nomarski
differential interference contrast microscope. Red-brown coloration revealed a positive reaction with the
sucrose transporter antibody. Arrows show the zones with the positive antibody reaction. e, epidermal
cells; en, endosperm; p, parenchymal cells; t, tracheidal elements; v, vascular tissue. Bar in A-C, E-H, 33
m; D, 160 m.
Figure 6. Medium acidification (A) and (B) ATP
hydrolysis and H+-pumping (C) mediated by
the PM H+ ATPase during embryo imbibition.
Changes in external pH were measured by
quenching of ACMA fluorescence
Germination starts when a seed is placed in water under favourable conditions,
and is completed when the radicle emerges from the embryo. Extensive amount of
information demonstrate the mobilization of complex carbohydrates associated
with the post-germinative phase, the growth of the seedling (Fig 1), but scarce
information are about the events associated with the mobilization of the embryo
endogenous soluble carbohydrates at early germination times, phases I and II of the
seed water uptake (Bewley & Black, 1994; Weitbrecht et al., 2011).
Contribution of the reserves of the embryo is overlooked in part because the
water uptake changes are higher after germination with the visible growing of the
aerial parts. But in order to sustain the moderate but essential metabolism of
embryo during the first hours of water uptake (Srivastava, 2002; Weitbrecht et al.,
2011) the embryo requires the use of its own reserves to provide the energy and
precursors for the synthesis of cell components and to sustain the radicle
emergence, the hallmark of germination. It has been described that radicle
extension involves cell division and/or cell elongation (Antipova et al., 2003), which
demands the incorporation of molecules that must be de novo synthesized and the
transport of nutrients from the outside to the embryo axes cells.
The aim of the present work was to understand the role of endogenous embryo
carbon reserves at early hours of maize germination and how the soluble sugars
are mobilized. In order to achieve the goals, the biological material used was the
embryo instead of the whole seed.
Imbibition for 0, 6, 12, 18, 24 and 30 h at 29C in 1% Agar
Figure 1 The time course of events associated with seed germination and subsequent
postgerminative seedling growth (Nonogaki, 2008).
Figure 2. Physiological parameters during maize embryo imbibition. (A) Time course of
germination (B) water and oxygen and uptake and (C) radicle elongation of embryos during
imbibition.
Imbibition
time (h)
Carbohydrate content
(mg carbohydrate embryo axis-1)
Carbohydrate content
(mg carbohydrate embryo-1)
Sucrose Glucose Fructose Sucrose Glucose Fructose
0 0.945 ± 0.282 0.108 ± 0.003 0.007 ± 0.001 9.092 ± 0.217 0.083 ± 0.002 0.006 ± 0.001
8 0.420 ± 0.064 0.046 ± 0.001 0.003 ± 0.003 5.446 ± 0.022 0.086 ± 0.034 0.025 ± 0.010
12 0.249 ± 0.023 0.075 ± 0.001 0.019 ± 0.005 4.272 ± 0.032 0.082 ± 0.050 0.015 ± 0.001
24 0.151 ± 0.014 0.039 ± 0.005 0.019 ± 0.006 1.486 ± 0.020 0.047 ± 0.005 0.034 ± 0.017
Table 1. Soluble carbohydrate levels in embryo and embryo-axis during germination.
Embryo and embryo axis were germinated at the indicated times and then carbohydrates
were extracted in ethanol and quantified by enzymatic coupled assays.
The left image is a schematic representation
of the components of the maize seed in a
longitudinal slice. Other images, longitudinal
views from embryos imbibed. Arrows show
that at 0 h the embryo has sucrose at the
scutellum (sc), and the patenchyma cells (scp).
At 8 h sucrose was low and located around
the embryo axis. Later (18 and 24 h), sucrose-
rich areas were found at the upper tip of the
embryo axes, in the plumula region (p), which
is starting to undergo cell growth to originate
the seedling leaves
Since the embryo axis does not undergo
growth and its main function is to provide
carbon and nitrogen backbones to the
embryo axis, which in turn do not have
enough reserve molecules and is engaged in a
program of active growth in germination
(Mayer and Poljakoff-Mayber, 1989). It is
possible that the scutellum protein and lipid
reserves (Fig 3B), and at minor extent its own
sucrose, may become a source of energy for
sugar transport to the embryo axes.
The high activity of the cell wall invertase
activity at the embryo (Fig 2B) suggests that
the embryo axis is actively using sucrose and
importing glucose.
Sucrose is the main carbohydrate at the embryo and embryo axis (Table 1). During the first 12 h,
sucrose was rapidly mobilized within the embryo and used by the embryo axis (Table 1). The
sucrose remaining in both tissues after 24 h was about 16% of the original reserves.
The large amounts of sucrose that diminished in the embryos throughout these 24 h were
apparently exported by the scutellum towards the embryo axes. To corroborate the temporal
disappearance we make the sucrose tissue localization (Fig 3A).
Non-imbibed embryos showed ability to
transport glucose and sucrose (Fig 4).
Glucose uptake by embryo and embryo
axes were 3 to 7-fold higher than the
sucrose uptake. Embryo axis glucose
uptake increases with the germination
time. In contrast, sucrose uptake was
lower and constant throughout the same
interval.
ZmSUT is a sucrose transporter that
could mediate the efflux of sucrose
(Carpaneto et al., 2005), in this case
from the scutellum to the embryo axis.
scutellum epidermis scutellum parenchyma cells,
scutellum vascular elements Vascular tracheidal elements
parenchymal cell of the plumule plumule Root cortex
CONCLUSI0N:The endogenous sucrose at the scutellum cells is discharged into
the symplast, and then moves through the vascular tissue to the
embryo axes cell apoplasts. There, sucrose is converted into
hexoses by the cell wall-invertases. Then, glucose and fructose
are translocated into the embryo axes cells by the hexose
transporter for nourishment and to drive radicle protrusion.
As the electrochemical H+ gradient
produced by the plasma membrane H+-
ATPase drives secondary transport and
apoplast acidification (Gaxiola et al.,
2007), we addressed the question
whether this enzyme was required to
promote the transport of the
carbohydrates in the scutellum and in
the embryo axes and produce the
medium acidification required for
radicle extension
CONCLUSION 2:The time-dependent expression of the plasma membrane H+-ATPase activity
correlates with the requirement of the generation of an electrochemical H+
gradient and acidification to support radicle elongation, which is in turn
associated to the mobilization of sugars from the scutellum to the embryo
axes by specific transporters.
This work was supported by the Universidad Nacional Autónoma de México (Grants PAPIIT IN207806, IN211409, IN203708, IN220511) and by the Consejo Nacional de Ciencia y Tecnología (CONACyT), México (55610, 101521).
OBJECTIVE
Immunolocalization of sucrose transporter was strong in: scutellum epidermis,
parenchyma cells, and vascular elements, and also in vascular tracheidal elements
at terminal development. Faint immunolabel is shown in parenchymal cell of the
plumule, top view of the plumule, epidermal and parenchymal cells of root cortex.
This differential ZmSUT1 localization suggested that the transporter was
predominantly expressed in cells that are involved in the flow of sucrose from the
scutellum symplast to the apoplastic spaces of the embryo axes.
Carbon mobilization at early maize embryo germination Sánchez-Nieto Sa, Sánchez-Linares La, Gavilanes-Ruíz Ma, Díaz-Pontones Db, Guzmán-Chávez Fa, Calzada-Alejo Va, Zurita-Villegas Va, Luna-
Loaiza Va, Moreno-Sánchez Rc.
a Dpto. de Bioquímica, Facultad de Química, Conjunto E. Universidad Nacional, Autónoma de México, Cd. Universitaria, Coyoacán. México 04510, D.F., México. bDpto. de Ciencias de la Salud, División de Ciencias Biológicas y de la Salud,Universidad Autónoma Metropolitana Iztapalapa. Apartado Postal 55535, México
09340, D.F., México. cDpto. de Bioquímica, Instituto Nacional de Cardiología Ignacio Chávez. Tlalpan, México 14080, D.F., México. E-mail: [email protected]
Maize embryos
Metabolite
extraction and
analysis
1. Carbohydrates
2. Tryglicerides
Enzyme obtention
and activity
determination
1. Hexokinase
II. Cell wall invertase
III. PM H+-ATPase
Localization of molecules
I. Sucrosehistochemistry
II. Immunohistochemistry
of transporters
Uptake detection
1. Sucrose and hexose (quantified by
liquid scintillation)
2. Oxygen
MATERIAL AND METHODSINTRODUCTION
RESULTSThe maize embryo germination is not affected by removing the endosperm (Fig
2A), and follows the typical triphasic curve of water and oxygen uptake, with the
two phases within the 0 and 24 h of imbibition (Fig 2 B). The radicle growth
was maximal about the 18 h (Fig 2C). Then for the subsequent experiments the
embryos were imbibed for 0, 6, 12, and 24 h.
Figure 3. Triglyceride content and the invertase
and hexokinase activities during maize embryo
germination. (A) Sucrose localization in embryos
imbibed at different times. Arrows indicate
sucrose-stained regions. (B) Cell wall-invertase
and hexokinase activities. (C) Triglyceride
content.Figure 4. Sucrose (A) and glucose uptake (B) by
embryo or embryo axes during imbibition (A).
Figure 5. Immunolocalization of the sucrose transporter in maize embryos. Embryos
were imbibed for 24 h, fixed, infiltrated in sucrose and frozen. Then, tissues were
sliced into 8 m sections and slices were processed for immunolocalization with the
sucrose transporter antibody and viewed in a Nomarski differential interference
contrast microscope. Red-brown coloration revealed a positive reaction with the
sucrose transporter antibody. Arrows show the zones with the positive antibody
reaction. e, epidermal cells; en, endosperm; p, parenchymal cells; t, tracheidal elements;
v, vascular tissue. Bar in A-C, E-H, 33 m; D, 160 m.
Figure 6. Medium acidification (A) and (B) ATP
hydrolysis and H+-pumping (C) mediated by
the PM H+ ATPase during embryo imbibition.
Changes in external pH were measured by
quenching of ACMA fluorescence
Figure 7. Immunolocalization of plasma membrane H+-ATPase in maize
embryos. Tissues seections were incubated with a plasma membrane
H+-ATPase antibody. Arrows show the positive reaction. c, cortex; cr,
cap root; ep, epidermis; r, root. Bar in A 500 m, B-D, 50m.
Germination starts when a seed is placed in water under favourable conditions, and is completed when the radicle
emerges from the embryo. Extensive amount of information demonstrate the mobilization of complex carbohydrates
associated with the post-germinative phase, the growth of the seedling (Fig 1), but scarce information are about the
events associated with the mobilization of the embryo endogenous soluble carbohydrates at early germination times,
phases I and II of the seed water uptake (Bewley & Black, 1994; Weitbrecht et al., 2011).
Contribution of the reserves of the embryo is overlooked in part because the water uptake changes are higher after
germination with the visible growing of the aerial parts. But in order to sustain the moderate but essential metabolism of
embryo during the first hours of water uptake (Srivastava, 2002; Weitbrecht et al., 2011) the embryo requires the use of
its own reserves to provide the energy and precursors for the synthesis of cell components and to sustain the radicle
emergence, the hallmark of germination. It has been described that radicle extension involves cell division and/or cell
elongation (Antipova et al., 2003), which demands the incorporation of molecules that must be de novo synthesized and
the transport of nutrients from the outside to the embryo axes cells.
The aim of the present work was to understand the role of endogenous embryo carbon reserves at early hours of maize
germination and how the soluble sugars are mobilized. In order to achieve the goals, the biological material used was the
embryo instead of the whole seed.
Imbibition for 0, 6, 12, 18, 24 and 30 h at 29C in 1% Agar
Figure 1 The time course of events associated with seed
germination and subsequent postgerminative seedling growth
(Nonogaki, 2008).
Figure 2. Physiological parameters
during maize embryo imbibition. (A)
Time course of germination (B) water
and oxygen and uptake and (C) radicle
elongation of embryos during
imbibition.
Imbibition
time (h)
Carbohydrate content
(mg carbohydrate embryo axis-1)
Carbohydrate content
(mg carbohydrate embryo-1)
Sucrose Glucose Fructose Sucrose Glucose Fructose
0 0.945 ± 0.282 0.108 ± 0.003 0.007 ± 0.001 9.092 ± 0.217 0.083 ± 0.002 0.006 ± 0.001
8 0.420 ± 0.064 0.046 ± 0.001 0.003 ± 0.003 5.446 ± 0.022 0.086 ± 0.034 0.025 ± 0.010
12 0.249 ± 0.023 0.075 ± 0.001 0.019 ± 0.005 4.272 ± 0.032 0.082 ± 0.050 0.015 ± 0.001
24 0.151 ± 0.014 0.039 ± 0.005 0.019 ± 0.006 1.486 ± 0.020 0.047 ± 0.005 0.034 ± 0.017
Table 1. Soluble carbohydrate levels in embryo and embryo-axis during germination.
Embryo and embryo axis were germinated at the indicated times and then carbohydrates
were extracted in ethanol and quantified by enzymatic coupled assays.
The left image is a schematic representation of the
components of the maize seed in a longitudinal
slice. Other images, longitudinal views from
embryos imbibed. Arrows show that at 0 h the
embryo has sucrose at the scutellum (sc), and the
patenchyma cells (scp). At 8 h sucrose was low and
located around the embryo axis. Later (18 and 24
h), sucrose-rich areas were found at the upper tip
of the embryo axes, in the plumula region (p),
which is starting to undergo cell growth to
originate the seedling leaves
Since the embryo axis does not undergo growth
and its main function is to provide carbon and
nitrogen backbones to the embryo axis, which in
turn do not have enough reserve molecules and is
engaged in a program of active growth in
germination (Mayer and Poljakoff-Mayber, 1989). It
is possible that the scutellum protein and lipid
reserves (Fig 3B), and at minor extent its own
sucrose, may become a source of energy for sugar
transport to the embryo axes.
The high activity of the cell wall invertase activity
at the embryo (Fig 2B) suggests that the embryo
axis is actively using sucrose and importing
glucose.
Sucrose is the main carbohydrate at the embryo and embryo axis (Table 1).
During the first 12 h, sucrose was rapidly mobilized within the embryo and used
by the embryo axis (Table 1). The sucrose remaining in both tissues after 24 h
was about 16% of the original reserves.
The large amounts of sucrose that diminished in the embryos throughout these
24 h were apparently exported by the scutellum towards the embryo axes. To
corroborate the temporal disappearance we make the sucrose tissue
localization (Fig 3A).
Non-imbibed embryos showed ability to transport
glucose and sucrose (Fig 4). Glucose uptake by
embryo and embryo axes were 3 to 7-fold higher
than the sucrose uptake. Embryo axis glucose
uptake increases with the germination time. In
contrast, sucrose uptake was lower and constant
throughout the same interval.
ZmSUT is a sucrose transporter that could
mediate the efflux of sucrose (Carpaneto et
al., 2005), in this case from the scutellum to
the embryo axis.
scutellum epidermis scutellum parenchyma cells,
scutellum vascular elements Vascular tracheidal elements
parenchymal cell of the plumule plumule Root cortex
Immunolocalization of sucrose transporter was
strong in: scutellum epidermis, parenchyma cells, and
vascular elements, and also in vascular tracheidal
elements at terminal development. Faint
immunolabel is shown in parenchymal cell of the
plumule, top view of the plumule, epidermal and
parenchymal cells of root cortex.
This differential ZmSUT1 localization suggested that
the transporter was predominantly expressed in
cells that are involved in the flow of sucrose from
the scutellum symplast to the apoplastic spaces of
the embryo axes.
CONCLUSION 1:The endogenous sucrose at the scutellum cells
is discharged into the symplast, and then
moves through the vascular tissue to the
embryo axes cell apoplasts. There, sucrose is
converted into hexoses by the cell wall-
invertases. Then, glucose and fructose are
translocated into the embryo axes cells by the
hexose transporter for nourishment and to drive
radicle protrusion.
As the electrochemical H+ gradient produced by
the plasma membrane H+-ATPase drives
secondary transport and apoplast acidification
(Gaxiola et al., 2007), we addressed the question
whether this enzyme was required to promote the
transport of the carbohydrates in the scutellum
and in the embryo axes and produce the medium
acidification required for radicle extension
A strong correlation was noted between the high
ATP hydrolysis activity and high medium
acidification at 18 h and 24 h (Fig. 5A, B). Within this
time-window, the phase II and phase III transition of
water uptake takes place (16 h) (Fig. 1B), i.e., the
time at which elongation of axial organs such as
plumule and mainly radicle occurs (Fig. 1C).
The rate and extent of acidification driven by the
H+-ATPase were higher at 12, 18 and 24 h as
compared to 6 h imbibition (Fig. 5C).
The H+-ATPase tissue distribution in the maize
embryo, showed a high staining level in the
plasmalemma of the root cells epidermis (Fig. 7A, B)
as well as in parenchymal cortex cells (Fig. 7C) of
the elongation and maturation zones of the root.
These results showed that the H+-ATPase was
present in regions involved in radicle growth
Root and cap root Root, transversal plane
Root cortex Control
CONCLUSION 2:The time-dependent expression of the plasma membrane H+-
ATPase activity correlates with the requirement of the
generation of an electrochemical H+ gradient and acidification
to support radicle elongation, which is in turn associated to
the mobilization of sugars from the scutellum to the embryo
axes by specific transporters.
This work was supported by the Universidad Nacional Autónoma de México (Grants PAPIIT IN207806, IN211409, IN203708, IN220511) and by the Consejo Nacional de Ciencia y Tecnología (CONACyT), México (55610, 101521).