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: sobeida@unam.mx

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: sobeida@unam.mx

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).