Botanical Studies (2008) 49: 19-24.

1

Dengwen Li and Liping Song contributed equally to this

study.

*

Corresponding author: E-mail: xthuang@public.tpt.tj.cn;

Tel: 022-23508874; Fax: 022-23508874.

INTRODUCTION

During the cell cycle, histones are subjected to a vari-

ety of post-translation modifications, including acetyla-

tion, methylation, phosphorylation, ubiquitylation, and

ribosylation. These different modifications can generate

synergistic or antagonistic interaction affinities for chro-

matin-associated proteins, which in turn dictate dynamic

transcriptionally silent chromatin states. Therefore, the

histone modifications and combinations represent a funda-

mental regulatory mechanism that has an impact on most

chromatin-templated processes, and many cellular process-

es (Jenuwein and Allis, 2001). The cell cycle-dependent

phosphorylation of histone H3 at serine10 (Ser-10 pH3)

has been certificated to be conserved in eukaryotes (Hend -

zel et al., 1997; Li et al., 2005; Wei et al., 1998). This post-

translational modification has been linked to transcription

activation (Thomson et al., 1999) during the interphase

and to chromosome condensation during mitosis (Van

Hooser et al., 1998; Wei et al., 1998).

A similar post-translational modification of H3 has also

been demonstrated for higher plants (Houben et al., 1999;

Kaszas and Cande, 2000; Yang et al., 2002). Using a site-

phosphorylation specific antibody, it was shown that in

wheat root tips Ser-10 pH3 started at early prophase and

vanished at telophase. The modification was concentrated

mainly in the pericentromeric regions at metaphase and

anaphase during cell division (Yang et al., 2002). The

function of the Ser-10 pH3 in wheat root cells is related to

the chromosome condensation during mitosis.

It has been shown that plants encode Aurora-like ki-

nases, analogous to the yeast aurora/lpl1 founding member

and the Aurora-related kinases of other organisms. In plant

cells, the Aurora-like kinase is responsible for phosphory-

lation of the histone H3 at serine10 (Demidov et al., 2005).

Some evidence has been published supporting the idea that

the mitotic phosphorylation and dephosphorylation of H3

are governed by IpI1/aurora kinase and Glc7/pp1 phospha-

tase in budding yeast and nematodes (Hsu et al., 2000). In

these models, both enzymes are required for H3 phosphor-

ylation and chromosome segregation. They are responsible

for the balance of H3 phosphorylation during mitosis in

Sacharomyces cerevisiae and Caenorhabditis elegans.

The hyperphosphorylation of the alfalfa cellular pro-

teins at low temperature has been shown to be caused by

differential sensitivity to cold between the protein kinase

and phosphatase by using a cell-free system of the plant

(Monroy et al., 1997). In the present study, immunofluo-

rescence microscopy and western blot were used to ana-

lyze the level of the Ser-10 pH3 in the freezing treatment

of wheat root cells. We found that the high level Ser-10

pH3 was present in the whole course of cell cycle in the

freezing treatment cells. This kind of post modification

of histone H3 involves a stringent reaction to the freezing

stress.

Freezing wheat root tips causes hyperphospharylation

of histone H3 at serine10 in the cell during mitosis

Liping SONG

1

, Dengwen LI

1

, Hao ZHOU, Ruming LIU, Cao KOU, Jiatong CHEN, and Xitai

HUANG*

Department of Biochemistry and Molecular Biology, NanKai University, Tianjin, 300071, P.R. China

(Received December 20, 2006; Accepted September 7, 2007)

ABSTRACT.

Plants are able to produce various responses to environmental changes. In this report, we dem-

onstrate that freezing of wheat root tips results in hyperphospharylation of Histone H3 at serine10. In normal

conditions, the serine10 phosphorylation of histone H3 occurs at the condensed chromosomes at prophase

and vanishes at telophase. The phosphorylated H3 is present mainly in the pericentromeric regions at meta-

phase and anaphase. However, in the frozen cells, the phosphorylation of histone H3 at serine10 is distributed

throughout the chromosome arms at all of the phases during mitosis, even at interphase and cytokinesis. The

results support the notion that hyperphospharylation of Histone H3 at serine10 is related to a stringent re-

sponse.

Keywords: Freezing; Hyperphospharylation; Immunofluorescence microscopy; Phosphorylation of histone H3

at serine10; Stringent response.

PHYSIOLOGY

20

Botanical Studies, Vol. 49, 2008

MATERIALS AND METHODS

Plant material

The wheat (Triticum aestivum cv. Chinese Spring) root

tips were taken from seeds germinating on wet filter paper

in Petri dishes at 25¢XC. The germinating seeds were main-

tained at -20¢XC for 1h for collecting the cold-treated wheat

root tips.

Specimen preparation

Root tips were fixed for 30 min in freshly prepared 4%

(W/V) paraformaldehyde (PFA, Sigma) solution contain-

ing phosphate-buffered saline (PBS, pH 7), washed for 45

min in PBS and digested at 37¢XC for 30 min in a mixture

of 2.5% (W/V) pectolyase Y-23 (Japan Yakult.) and 2.5%

(W/V) cellulase (Japan Cal-Bio) dissolved in PBS. Root

tips were washed 15 min in PBS and squashed between

a glass slide and a coverslip in 45% (V/V) acetic acid.

After being frozen in liquid nitrogen, the coverslips were

removed, and the slides were transferred immediately into

PBS.

Immunofluorescence staining

Slides were permeabilized with 0.5% Triton X-100 in

PBS for 30 min at room temperature (RT). To avoid non-

specific antibody binding, Slides were blocked for 30 min

in 4% (w/v) bovine serum albumin (BSA) in PBS at RT

prior to two washes in PBS for 5 min each and incubated

with the primary antibodies in a humid chamber. Poly-

clonal affinity purified rabbit antibody against histone H3

phosphorylated at serine10 (Upstate Biotechnology, cata-

log no. 06-570) and mouse monoclonal antibody against

microtubules (Zymed Laboratories Inc., catalog no.

32-2500) were diluted 1:400 in PBS with 3% BSA. After

12 h incubation at 4¢XC and washing for 15 min in PBS,

the slides were incubated in FITC-conjugated anti-rabbit

IgG (Upstate Biotechnology, catalog no. 02-15-06) and

TRITC-conjugated anti-mouse IgG (Upstate Biotechnol -

ogy, catalog no. 1090-03) diluted 1:200 in PBS, 3% BSA

for 1 h at 37¢XC. After final washes in PBS, the preparations

were mounted in DAPI as counterstain.

Confocal laser microscopy

Confocal scanning microscopy was performed using

a TCS-NT Leica microscope (Lasertechnik, Heidelberg,

Germany); an argon-krypton ion laser was adjusted at an

excitation wavelength of 345 nm, 488 nm, and 568 nm.

Fluorescent images were captured in sequential mode. Se-

rial optical sections were taken. Selected paired sections

were then processed to produce single composite, color-

merged overlay images.

Plant histone extraction and Western blot

analysis

We collected the same weight of normal/freezing wheat

root tips for analysis. Histone-enriched protein extracts

were prepared essentially as described (Yu et al., 2004).

Briefly, the wheat tips were collected and ground in liquid

N

2

into fine powder. After homogenization of the powder

in buffer A (0.4 M sucrose, 10 mM Tris-HCl pH 8.0, 10

mM MgCl

2

, 0.1 mM PMSF, 5 mM £]-mercaptoethanol)

(approximately 5 ml buffer per gram of powder), the re-

sulting slurry was filtered through 200 and 100 lm nylon

meshes. The filtrate was centrifuged at 1,500 g for 20 min.

The pellet was washed in buffer B (0.25 M sucrose, 10

mM Tris-HCl pH 8.0, 10 mM MgCl

2

, 1% Triton X-100, 0.1

mM PMSF, 5 mM £]-mercaptoethanol), then homogenized

in 1 ml buffer C (1.7M sucrose, 10 mM Tris-HCl pH

8.0, 2 mM MgCl

2

, 0.15% Triton X-100, 0.1 mM PMSF,

5mM £]-mercaptoethanol). The chromatin was treated

twice for 45 min with 0.4 M H

2

SO

4

, and the proteins were

precipitated with 5 volumes of ethanol for 48 h at -20¢XC.

After centrifugation at 12,000 g for 10 min, the pellet was

washed with 80% ethanol, dried, and resuspended in 0.01N

HCl. The samples were resolved on 15% SDS-PAGE and

transferred onto PVDF membrane (Amersham Bioscienc-

es). The rabbit anti-phospho-Histone H3 (Ser-10) (Upstate

Biotechnology, catalog no. 06-570), diluted 1:1000, was

used to detect the target protein. Membranes were exposed

to a horse radish peroxidase (HRP)-conjugated goat anti-

rabbit IgG polyclonal antibody (Upstate Biotechnology,

catalog no. 474-1506, 1:5000 dilution). A DAB detection

system was used for protein detection and visualized in a

UVP Bio-imaging system.

RESULTS

Dynamic distribution of Ser-10 pH3 in wheat

cell during mitosis

To determine the localization of Ser-10 pH3, microtu-

bules and DNA in the cell during mitosis, a three-color

immunofluorescence label was used in this work. At pre-

prophase (Figure 1A), the green signal of Ser-10 pH3

cannot be detected in the cell, but a red preprophase band

composed of microtubule is present in middle of the cell

surrounding by the inner-cytoplasmic membrane. At pro-

phase; the green immunofluorescence signal of the Ser-10

pH3 appeared and spread along with the condensed chro-

mosomes in the nuclei. This indicated that the phosphory-

lation of H3 at serine10 was global at this phase of mitosis

in wheat cells (Figure 1B). At metaphase, the chromo-

somes line up at the equatorial plate of the cell and interact

with the microtubules which arrange themselves in paral-

lel perpendicular to the equator. The green labeling signal

(Ser-10 pH3) was vivid mainly in the pericentromeric

regions while it appeared weak on the chromosomal arms

during the metaphase and anaphase (Figure 1C, D). The

chromosomes began to decondense, and the green labeling

signal was weak until it disappeared at telophase and cyto-

kinesis (Figure 1E, F). This indicates that the distribution

of the serine10 phosphorylation of histone H3 changes fol-

lowing the progression of mitosis.

In wheat root tip cells, microtubules show dynamic

structural changes during cell cycle progression. The corti-

SONG et al. ¡X Freezing causes hyperphospharylation in plant cell

21

cal microtubules, preprophase band, spindle microtubules,

and phragmoplast, all of which are plant specific microtu -

bules structures, can be observed from the late G2 phase

through to telophase (Figure 1).

Abnormal distribution of Ser-10 phospharylated

histone H3 in freezing wheat cell

The wheat root tips were freezing at -20¢XC for 1 h

before the cell specimen preparation and indirect immu-

nofluorescence staining. It is interesting that the dynamic

distribution of Ser-10 pH3 was altered by cold treatment.

The Ser-10 pH3 is distributed throughout the chromosome

arms during mitosis, even in interphase and cytokinesis

(Figure 2A, F). The regular dynamic distribution of Ser-10

pH3 cannot be found in the plant cell, but the organization

of microtubules in freezing wheat cells is the same as in

normal cells mostly at different phase (Figure 2A-F). Two

hundred of the frozen mitotic cells were examined by con-

Figu re 1. The dynamic distribu-

tion of the Ser-10 pH3 in wheat

cell during mitos is. Ser-10 pH3

was labeled with FITC (gree n);

£\-tubulin and DNA were labeled

wi t h T R IT C ( re d ) a nd D AP I

(blue), respectively. A: pre-pro-

phase; B: prophase; C: metaphase;

D: anaphase; E: telophase; F: cy-

tokinesis.

22

Botanical Studies, Vol. 49, 2008

focal microscopy; all of them showed irregularly strong

immunofluorescence staining. Two important different

points between frozen and untreated wheat tip cell are:

1) the strong phosphorylation signal appears at all phases

from preprophase (Figure 2A, with a preprophase band in

the cell) to telophase (Figure 2F, the phragmoplast formed

in); 2) the Ser-10 pH3 appears all along the chromosome,

not only the pericentromeric region. The results indicate

that freezing treatment causes global phosphorylation of

histone H3 at Ser-10 in mitotic wheat cells and disturbs

regular distribution of the modified histone. The cold treat-

ment does not, however, destroy the constructions of mi-

crotubule that are mainly found in the cell.

The histones extracted from normal/cold treated

cells were analyzed by SDS-PAGE and western

blot

The histones of normal/cold treated cells were extracted

Figure 2. Abnorm al dis tribution of

Ser-10 pH3 in freezing wheat cell dur-

ing mitosis. Ser-10 pH3 were labeled

wi th F IT C (gre en); £\-tu bulin and

DNA were labeled with TRITC (red)

and DAPI (blue), respectively. A: pre-

prophase; B: prophase; C: metaphase;

D: anaphase; E: telophase; F: cytoki-

nesis.

SONG et al. ¡X Freezing causes hyperphospharylation in plant cell

23

according to "Material and Methods." The equal amounts

of the two samples were resolved on 15% SDS-PAGE, and

the core histones H2A, H2B, H3, and H4 bands lined up

on the markers range of molecular weight 10.0 kDa-28.0

kDa (Figure 3a). The levels of the Ser-10 pH3 in cold-

treated samples are much higher than those of the normal

samples, as shown in analysis of the western blot (Figure

3b). These results are consistent with that of the immuno-

fluorescence microscopy.

DISCUSSION

Ser-10 pH3 appeared in the eukaryotic cells as a mito -

sis marker during the cell cycle. It has been reported that

these modified proteins are related to the chromosome

condensation during mitosis (Van Hooser et al., 1998;

Houben et al., 1999; Yang et al., 2002; Li et al., 2005). By

using indirect immunofluorescence labeling and laser con -

focal microscopy, Ser-10 phosphorylation of histone H3

is proven to occur at prophase in the whole chromosomes

of wheat cell root tips. Then, the phosphorylation signal is

concentrated at the pericentromeric region in punctuate at

metaphase and anaphase before disappearing at telophase.

The dynamic distribution of Ser-10 phosphorylated his-

tone H3 suggests that the modified protein at prophase and

premetaphase may serve to promote chromatin condensa-

tion; and the Ser-10 pH3 is collected in pericentromeric

region at metaphase, meaning that the Ser-10 pH3 may

be involved in active kinetochore and direct the daughter

chromosomes moving to the two poles of the dividing cell

successfully. The distribution and function of Ser-10 pH3

at metaphase and anaphase is quite different in plants and

mammals. The modified histone has been proved to be

collected in the centre of the spindle at metaphase and to

be involved in the midbody. The Ser-10 pH3 plays a role

in cytokinesis in mammalian cells (Li et al., 2005; Song et

al., 2007).

In the present study, we found that freezing causes

hyperphosphorylation of histone H3 at Ser-10 in wheat

root tip cells during mitosis. The distribution of Ser-10

pH3 in the cell is neither the same as in normal cells (as

described above) nor the same as in ice-water treated cells

(Manzanero et al., 2002). The modified protein is present

at all of the phases from preprophase to cytokinesis in the

treated cells. The hyperphosphorylation of histone H3 in

the frozen cell was demonstrated by SDS-PAGE and west-

ern blot of acid-extracted histone samples.

The hyperphosphorylation of H3 in wheat mitotic cell

caused by freezing might be due to destruction of the bal-

ance between aurora-like kinases and phosphorylatase, if

the phophatase is also sensitive to cold stress.

As demonstrated above, the Ser-10 pH3 disappears

in the later phases of the mitotic wheat cell. This means

that the activity of aurora kinase has fallen to a low level.

The Ser-10 pH3 is confined to the pericentrimeric region

at a late phase of the mitotic cell, so the irregular hyper-

phosphorylation of histone caused by freezing treatment

resembles an initial regulating reaction of the cell. As the

plant cell experiences freezing stress, it may conduct a

stringent response against the severe conditions, and some

kinases in the cell would activate histone phosphoryla-

tion and cause chromatin condensation. The cell reduces

the metabolite level of the cell in order to survive the low

temperature. How freezing induces the hyperphospharyla-

tion of histone at Ser-10 should be a subject of intensive

research.

LITERATURE CITED

Demidov, D., D. Van Damme, D. Geelen, F.R. Blattner, and A.

Houben. 2005. Identification and dynamics of two classes

of aurora-like kinases in Arabidopsis and other plants. Plant

Cell 17: 836-848.

Hendzel, M.J., Y. Wei, M.A. Mancini, A. Van Hooser, T. Ranalli,

B.R. Brinkley, D.P. Bazett-Jones, and C.D. Allis . 1997.

Mitosis-specific phosphorylation of histone H3 initiates pri-

marily within pericentromeric heterochromatin during G2

and spreads in an ordered fashion coincident with mitotic

chromosome condensation. Chromosoma 106: 348-360.

Houben, A., T. Wako, R. Furushima-Shimogawara, G. Presting,

G. Kunzel, I.I. Schubert, and K. Fukui. 1999. Short commu-

nication: the cell cycle dependent phosphorylation of his-

tone H3 is correlated with the condensation of plant mitotic

chromosomes. Plant J. 18: 675-679.

Hsu, J.Y., Z.W. Sun, X. Li, M. Reuben, K. Tatchell, D.K. Bish-

op, J.M. Grushcow, C.J. Brame, J.A. Caldwell, D.F. Hunt,

R. Lin, M.M. Smith, and C.D. Allis. 2000. Mitotic phos-

phorylation of histone H3 is governed by Ipl1/aurora kinase

and Glc7/PP1 phosphatase in budding yeast and nematodes.

Cell 102: 279-291.

Jenuwein, T. and C.D. Allis. 2001. Translating the histone code.

Science 293: 1074-1080.

Kaszas, E. and W.Z. Cande. 2000. Phosphorylation of histone

H3 is correlated with changes in the maintenance of sister

chromatid cohesion during meiosis in maize, rather than

Figure 3. The SDS-PAGE (a) and Western blot (b) of the histone

extracted from the normal/cold treated cells of the wheat root

tips. M: Molecular weight marker; N: Histones of the normal

cells; C: Histones of the cold-treated cells.

24

Botanical Studies, Vol. 49, 2008

the condensation of the chromatin. J. Cell Sci. 113(Pt 18):

3217-3226.

Li, D.W., Q. Yang, J.T. Chen, H. Zhou, R.M. Liu, and X.T.

Huang. 2005. Dynamic distribution of Ser-10 phosphory-

lated histone H3 in cytoplasm of MCF -7 and CHO cells

during mitosis. Cell Res. 15: 120-126.

Manzanero, S., T. Rutten, V. Kotseruba, and A. Houben. 2002.

Alterations in the distribution of histone H3 phosphoryla-

tion in mitotic plant chromosomes in response to cold treat-

m ent and the protein phosphatase inhibitor cantharidin.

Chromosome Res. 10: 467-476.

Monroy, A.F., E. Labbe, and R.S. Dhindsa. 1997. Low tempera-

ture perception in plants: effects of cold on protein phos-

phorylation in cell-free extracts. FEBS Lett. 410: 206-209.

Song, L., D. Li, R. Liu, H. Zhou, J. Chen, and X. Huang. 2007.

Ser-10 phosphorylated histone H3 is involved in cytokinesis

as a chromosomal passenger. Cell Biol. Int. 31: 1184-1190.

Thomson, S., L.C. Mahadevan, and A.L. Clayton. 1999. MAP

kinase-mediated signalling to nucleosomes and immediate-

early gene induction. Semin. Cell Dev. Biol. 10: 205-214.

Van Hooser, A., D.W. Goodrich, C.D. Allis , B.R. Brinkley,

and M.A. Mancini. 1998. Histone H3 phosphorylation is

required for the initiation, but not maintenance, of mam-

malian chromosome condensation. J. Cell Sci. 111(Pt 23):

3497-3506.

Wei, Y., C.A. Mizzen, R.G. Cook, M.A. Gorovsky, and C.D.

Allis. 1998. Phosphorylation of histone H3 at serine 10 is

correlated with chromosome condensation during mitosis

and meiosis in Tetrahymena. Proc. Natl. Acad. Sci. USA 95:

7480-7484.

Yang, Q., X.T. Huang, Z.H. Geng, and X.D. Yu. 2002. Localiza-

tion of phosphorylated histone H3 at mitosis and meiosis in

wheat. Acta Bot. Sin. 44: 1403-1408.

Yu, Y., A. Dong, and W.H. Shen. 2004. Molecular characteriza-

tion of the tobacco SET domain protein NtSET1 unravels

its role in histone methylation, chromatin binding, and seg-

regation. Plant J. 40: 699-711.