Annals of Botany 101: 277–283, 2008
doi:10.1093/aob/mcm089, available online at www.aob.oxfordjournals.org
Possible Role of Pectin-containing Mucilage and Dew in Repairing Embryo DNA
of Seeds Adapted to Desert Conditions
Z HE N Y IN G HU A NG 1 , IVA N B O U B R I A K 2, 3 , DA P HN E J . O S B O R N E 4 , M IN G DO N G 1 AND
Y I T Z C H A K G U T T E R M A N 5, *
1
Laboratory of Quantitative Vegetation Ecology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, P.R.
China, 2Institute of Cell Biology and Genetic Engineering UAS, Kiev, 03143, Ukraine, 3Department of Biochemistry,
Oxford University, South Parks Road, Oxford OX1 3QU, UK, 4Oxford Research Unit, The Open University, Foxcombe
Hall, Boars Hill, Oxford OX1 5HR, UK and 5Jacob Blaustein Institute for Desert Research and Department of Life
Sciences, Ben-Gurion University of the Negev, Sede Boker Campus, 84990, Israel
Received: 31 January 2007 Returned for revision 22 February 2007 Accepted: 9 March 2007 Published electronically: 11 May 2007
† Background and Aims Repair of damage to DNA of seed embryos sustained during long periods of quiescence
under dry desert conditions is important for subsequent germination. The possibility that repair of embryo DNA
can be facilitated by small amounts of water derived from dew temporarily captured at night by pectinaceous
surface pellicles was tested. These pellicles are secreted during early seed development and form mucilage when
hydrated.
† Methods Seeds of Artemisia sphaerocephala and Artemisia ordosica were collected from a sandy desert. Their
embryos were damaged by gamma radiation to induce a standard level of DNA damage. The treated seeds were
then exposed to nocturnal dew deposition on the surface of soil in the Negev desert highlands. The pellicles
were removed from some seeds and left intact on others to test the ability of mucilage to support repair of the
damaged DNA when night-time humidity and temperature favoured dew formation. Repair was assessed from fragmentation patterns of extracted DNA on agarose gels.
† Key Results For A. sphaerocephala, which has thick seed pellicles, DNA repair occurred in seeds with intact pellicles after 50 min of cumulative night dew formation, but not in seeds from which the pellicles had been removed.
For A. ordosica, which has thin seed pellicles, DNA repair took at least 510 min of cumulative night dewing to
achieve partial recovery of DNA integrity. The mucilage has the ability to rehydrate after daytime dehydration.
† Conclusions The ability of seeds to develop a mucilaginous layer when wetted by night-time dew, and to repair
their DNA under these conditions, appear to be mechanisms that help maintain seed viability under harsh desert
conditions.
Key words: Adaptation strategy, Artemisia, desert dew, DNA repair, pectin, pellicle, seed survival.
IN TROD UCT IO N
The ability of seeds of most temperate species to cease DNA
replication in the embryo and then undergo a subsequent
dehydration to less than 5 % moisture content with no
permanent cellular damage or genomic impairment permits
long-term seed survival for decades or even centuries
(Osborne, 1980, 2000). Water is a pre-eminent requirement
for metabolic re-activation and germination. If the water potential is too negative to initiate full germination, seeds can still
initiate certain biochemical processes that allow embryo
cells to progress to the S1 stage of the first cell cycle. Such
seeds will undergo important processes of pre-germination,
including those crucial for maintaining their viability, for
example DNA repair (Elder et al., 1987; Boubriak et al.,
2001). These processes can improve subsequent performance
under suboptimal conditions for germination. For this reason,
their induction is widely practised by industry. It is known as
‘priming’ or ‘preconditioning’ because the embryos remain
tolerant to desiccation and can be re-dried without harm in
the short-term (Heydecker et al., 1973; Heydecker, 1977;
Heydecker and Coolbear, 1977).
* For correspondence. E-mail [email protected]
A related phenomenon may apply to seeds of many
annuals and shrubs of desert regions that have evolved
special survival mechanisms. A limited number of species
have adapted to shifting dunes or to the more stable
sands, such as those to be found in the Mu-Us Desert in
China or in the Negev Desert of Israel. These species
have attracted attention because of their potential for undergoing opportunistic DNA repair as part of their long-term
survival strategy that necessarily involves survival for
many years on or near the surface of dry sand and
exposed to intensive UV irradiation. In such dehydrated
conditions, the embryos of dry seeds slowly degenerate,
enzyme proteins lose function and, in particular, the DNA
of nuclear and mitochondrial genomes suffer progressive
cleavage and other in situ damage from non-enzymatic
methylation, oxidation and dimerization of nucleotides
(Osborne and Boubriak, 1994; Osborne, 2000). They can
also suffer from random fragmentation of chromosomal
sequences (Cheah and Osborne, 1978). Unless the activity
of the enzymes of the DNA repair complex (a and b polymerases, DNA ligase) (Elder et al., 1987; Osborne et al.,
2002) remain sufficiently functional before germination is
fully initiated, the embryo cells will fail to recover a functional genome and the seed dies. The maintenance of an
# The Author 2007. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: [email protected]
278
Huang et al. — Pectin Pellicles, Dew and Seed Embryo DNA Repair
operational DNA repair complex is therefore an essential
requirement for long-term survival in the dry state.
Experiments were conducted linking repair of DNA to
the presence of a water-absorbing polysaccharide pellicle
deposited on the outside of the seeds (achenes) of
Artemisia sphaerocephala, an important pioneer plant
occurring in moving sand dunes (Wei and Tu, 1980) and
of Artemisia ordosica, a species restricted to stable sands
(Huang and Gutterman, 1999a, 2000). The pellicles form
a mucilaginous layer after wetting (e.g. by dew forming at
night when temperatures fall markedly in the desert). The
findings indicate that this night-time trapping of water is
sufficient to support repair to damaged DNA even though
hydration levels are too low for germination. In this way,
the integrity of the genome can be retained over the long
term (Elder and Osborne, 1993). It is known that as little
as 30 min is sufficient for repair of accumulated DNA
lesions to take place (Boubriak et al., 1997). Here the
extent to which hydrated mucilage can maintain genome
integrity during dew formation was examined under
actual night-time desert conditions and before the seed is
again dehydrated in the hot desert daytime air.
M AT E R I A L S A N D M E T H O D S
Location of seed collection
Artemisia sphaerocephala and A. ordosica flower from July
to August. During development, achenes are protected by
the inflorescences and thus protected from wetting by rain
until after dispersal. The achenes begin to mature from
mid September to early November and are subsequently
dispersed by wind and adhere to sand grains by the mucilaginous layer that forms on the achene surface when wetted.
Mature achenes of A. sphaerocephala and A. ordosica
were collected from dry unopened inflorescences in
December, 2002, from natural populations in the Mu-Us
sandy desert in Yulin (388060 N, 1078300 E; 1288 m a.s.l.),
China. Here the annual rainfall varies from 100 to
450 mm and falls mainly from July to September with
most night-time dew deposition occurring in August and
September (Zhang, 1986, 1994). In the laboratory, inflorescences were manually shaken to detach the achenes, which
were stored in a closed cotton bag at 4 8C.
Following irradiation, all embryos examined showed substantial damage to DNA as assessed by alkaline DNA electrophoresis (Elder and Osborne, 1993), which then became
the basis for setting-up DNA repair assessments in vivo.
Dew experiment in the desert
These experiments were made at the Jacob Blaustein
Institute for Desert Research, Ben-Gurion University, in
the Negev Desert highlands of Israel (308510 N, 348460 E;
460 m a.s.l.) where annual rainfall varies from 29 to
165 mm and occurs mainly from October to April
(Gutterman, 2002). Night-time dew deposition is most frequent from July to October (Zangvil, 1996). Dew deposition conditions in the Negev are similar to those found
in the shifting sand deserts in China (Zhang, 1986;
Kidron, 2005).
After radiation treatment, two replicated samples (50 per
set) of cleaned and intact lots of A. sphaerocephala seeds
and of intact A. ordosica seeds were weighed and spaced
out individually on the surface of Negev desert sand previously rinsed with distilled water. The weighed sand was
held in Petri dishes to a depth of 3 mm. The Petri dishes
were arranged on trays and the edges of trays were protected
by sticky glue against predators during nightly exposures to
high humidity and dew deposition. These experiments were
carried out in the field between 2000 h and 1000 h from 24
July to 2 August, 2003. The dishes were set up at the same
time each evening for nine nights. From 0500 h on the next
morning (after the time of maximum dew deposition), trays
were repeatedly returned to the laboratory for repeated
re-weighings every 15 min to determine rates of water
loss while on the desert floor. Weighings were continued
until the seeds had returned to their original air-dry
weight (Fig. 1). After final weighing, the seeds were
returned to the desert. Empty dishes were used to take
account of any changes in sand or dish weights, although
none proved large enough to affect the results.
Not all nights were dew nights, so some sets of seeds
were left out on consecutive nights to accumulate a
period of time of .90 % relative humidity (RH) in the
external air. These were the conditions when dew formed
Pellicle removal and gamma irradiation
A quantified dose of gamma radiation was used to induce
the same amount of single-strand DNA breaks into embryo
DNA (Elder and Osborne, 1993; Boubriak et al., 1997).
Prior to this, pellicles were removed from half the seeds
of A. sphaerocephala as follows. The seeds are small
(1.0 – 1.5 mm long) and were held in water for exactly
0.5 h and then rubbed rapidly on filter paper to remove
the pellicle as confirmed under the light microscope.
Batches of cleaned and intact A. sphaerocephala seeds
and intact seeds of A. ordosica were then gamma-irradiated
(1000 Gy) using a 137Cs, RX 30/55 M Irradiator (Gravetom
Industries, Gosport, Hampshire, UK) to induce a standard
amount of damage to the DNA of embryo cells.
F I G . 1. Time-course data of morning water loss by 50-mg samples of
seeds of Artemisia sphaerocephala (with or without pellicles) and
Artemisia ordosica from the time of maximum night hydration by dew
until the samples regained their original weight with the early morning
rise in temperature. Seeds were kept on the desert floor during drying.
An example is shown of one of 11 dew-loss measurements.
Huang et al. — Pectin Pellicles, Dew and Seed Embryo DNA Repair
279
in the desert. These accumulated periods of dew ranged
from 50 to 390 min. Throughout these procedures, the
monitoring station of the Desert Meteorology Unit of
Jacob Blaustein Institute recorded relative humidity
(Fig. 2) and temperature. Temperature and relative humidity
measurements were taken every 10 min via a thermohygrograph (W. Lambrecht GmbH Gottingen, Germany). Data
recording software was prepared by ‘Nimbus’, Israel.
After the night-time exposures and once air dry, all the
weighed individual sets of seeds were enclosed in
Eppendorf tubes for DNA fragmentation analysis.
DNA extraction and DNA fragmentation analyses
After the last desert exposure, the dry seeds were
analysed for DNA fragmentation patterns. In this way the
extent of any repair of the original irradiation-induced
DNA damage was determined. Two replicates of each
weighed sample of A. sphaerocephala seeds (70 mg with
pellicle; 50 mg minus pellicle) and 60 mg of A. ordosica
seeds were each ground in a mortar with three successive
additions of dry ice. The resulting powder was extracted
by using the Wizard Genomic DNA Kit (Promega Corp.,
Madison, WI, USA) as recommended by the manufacturer,
except that nuclei lysis solution was used throughout for
extraction. The DNA extracts were purified by
re-suspension and ethanol precipitation and quantified by
micro-cell UV analysis at 260 nm and by Pico GreenTM
(Molecular Probes Europe, Breda, the Netherlands)
fluorescence.
Equal
DNA
loadings,
2 mg
for
A. sphaerocephala and 4 mg for A. ordosica, were applied
to 0.8 % agarose gels and subjected to electrophoresis at
55 mA for 45 min. DNA was visualized with ethidium
bromide and the gels scanned and recorded on a UVP
data image analyser (Spring Scientific Ltd, New York,
USA) (Figs 3 and 4).
F I G . 3. Mobility scans of DNA extracted from seeds of Artemisia
sphaerocephala following gamma-irradiation treatments of 1000 Gy
when dry and then an exposure to monitored periods of night-time
with dew deposition in the desert of the Negev. Lane 1, DNA marker,
125– 23130 bp; Lane 2, dry seeds, pellicle removed; Lane 3, dry seeds,
pellicle removed, irradiated; Lane 4, dry seeds, pellicle intact; Lane 5,
dry seeds, pellicle intact, irradiated; Lane 6, seeds with pellicle
removed after five cold desert nights (no dew); Lane 7, seeds with pellicle intact and after five cold desert nights (no dew); Lane 8, seeds with
pellicle removed and after 50 min of night-time dew deposition; Lane 9,
seeds with pellicle intact and after 50 min of night-time dew deposition;
Lane 10, seeds with pellicle removed and after 120 min of night-time
dew deposition; Lane 11, seeds with pellicle intact and after 120 min
of night-time dew deposition; Lane 12, seeds with pellicle removed
and after 390 min of night-time dew deposition; Lane 13, seeds with pellicle intact and after 390 min of night-time dew deposition; Lane 14,
DNA marker, 500–5000 bp.
Scanning electron microscopy
Dry achenes or achenes that had been exposed to 340 min
of one night dew and dried to their original weight were
examined for structural changes following hydration of
F I G . 2. Daily rhythm of relative humidity of the air of the Negev desert
during exposure of seeds of Artemisia sphaerocephala and Artemisia ordosica on the desert floor. Total time of dew deposition (i.e. duration above
90 % of relative humidity in minutes) is shown on the three nights when
dew was deposited on the seeds.
F I G . 4. Mobility scans of DNA extracted from seeds of Artemisia
ordosica following gamma-irradiation treatments of 1000 Gy when dry
and then an exposure to monitored periods of night-time with dew deposition in the Negev desert. All pellicles were intact. Lane 1, DNA
marker, 1000– 12 000 bp; Lane 2, intact dry seeds; Lane 3, irradiated dry
seeds; Lane 4, seeds exposed to 50 min of night-time dew deposition;
Lane 5, seeds exposed to 120 min of night-time dew deposition; Lane 6,
seeds exposed to 390 min of night-time dew deposition; Lane 7, seeds
exposed to 510 min of night-time dew deposition; Lane 8, DNA marker,
100–1500 bp.
280
Huang et al. — Pectin Pellicles, Dew and Seed Embryo DNA Repair
F I G . 5. Scanning electron micrographs of sections of seeds of Artemisia sphaerocephala (A, C) and Artemisia ordosica (B, D). A and B show dry seeds
at the time of shedding. C and D show seeds after exposure to 340 min of dew deposition and subsequent re-drying. The pellicle layer of
A. sphaerocephala is shown to collapse when dried after wetting with dew (C). This does not affect its ability to reform mucilage on further re-wetting.
P, pellicle; T, testa; Cot, cotyledonary cells.
the pellicle and later re-drying. Achenes of
A. sphaerocephala and A. ordosica were first halved transversely with a sharp scalpel to expose a cross-section of the
pellicles (Fig. 5). For scanning electron microscopy,
material was mounted on grids and coated with gold in a
gold sputter at reduced pressure. Specimens were then
examined and photographed in a Zeiss SUPRA 55VP
(Carl Zeiss Ltd, Jena, Germany) analytical scanning electron microscope.
R E S U LT S
Repeated weighings of duplicate sets of seeds, set out on
Negev sand, in weighed Petri dishes allowed estimations
of maximum night-time dew deposition on the seeds as
well as the rates of drying through the early morning until
the seeds regained their original weight. The experiments
were performed over 10 d, which included some nights
when no dew was formed although temperatures decreased.
Only dew deposition resulted in pellicles swelling to form
a mucilaginous covering. Without pellicle, seeds of
A. sphaerocephala returned to the original weight within
1.5 h of dewing, whereas those with intact pellicles took
at least 3 – 4 h (Fig. 1) indicating a larger water-carrying
capacity of seeds with pellicles in place. Seeds of
A. ordosica, which have only thin pellicles, took only 2 h
to dry back to their normal daytime weight.
Figure 2 shows relative humidity during the Negev
experiments. Dew deposition took place during three
nights out of nine when there was no wind and
clear skies and continued as long as relative humidity was
90 %. The sum of the exposures to dew-forming conditions were 0, 50 (one night’s dew), 120 (one night’s
dew), 390 (two nights’ dew: 50 min þ 340 min) and 510
(three nights’ dew: 50 min þ 120 min þ 340 min) min.
Seeds with dew condensation could easily be recognized
by a glistening of the hydrated mucilage.
Mobility scans of extracted DNA (Fig. 3) show that the
gamma-irradiation of dry seeds effectively fragmented
embryo DNA as indicated by a long smear (Lane 3).
However, only 50 min of dew was needed to permit some
repair of this damage (short smear in Lane 9) in seeds of
A. sphaerocephala with intact pellicles. Repair was more
complete when dew deposition was extended to 120
(Lane 11) or 390 min (Lane 13). If the pellicle was
removed, little or no repair occurred (Lanes 8, 10 and 12)
with the long smears indicating extensive DNA fragmentation that could not be rescued by dew deposition. In comparison with those for A. sphaerocephala, scans of DNA
from intact A. ordosica seeds (Fig. 4) revealed that DNA
repair of gamma-irradiation-induced fragmentation takes
much longer. At least 510 min of cumulative night-time
with dew was needed to achieve even partial recovery of
DNA integrity (Lane 7). This is in line with the relatively
thin pellicular covering of this species and its corresponding
lower water-retaining capacity.
Scanning electron micrographs (Fig. 5) illustrate how the
pellicle covering is deposited during seed formation, the
pellicle layer of A. ordosica being about one-third the thickness of that of A. sphaerocephala (see also Huang and
Huang et al. — Pectin Pellicles, Dew and Seed Embryo DNA Repair
Gutterman, 2000). In addition, the pellicle structure of
A. sphaerocephala is shown to collapse after exposure to
dew and subsequent dehydration. However, these collapsed
pellicles were able to reform the mucilage layer when
re-wetted.
DISCUSSION
Some of the most common annual and perennial plant
species
occurring in the Saharo-Arabian and
Irano-Turanian phytogeographical regions of the Negev
desert of Israel (Feinbrun-Dothan, 1978, 1986; Zohary,
1966, 1972) have seeds which develop a mucilaginous
layer on their outer surface shortly after wetting
(Gutterman, 1993, 2002). The mucilage is generated from
pectin-rich pellicles on the testa or pericarp layers of oneseeded fruits or seeds (Zohary, 1962). The various species
have different strategies and mechanisms of seed dispersal
and germination. Some have their seeds dispersed by
wind at the beginning of the summer and others by rain
(Gutterman et al., 1967; Baskin and Baskin, 1998;
Gutterman, 2002). In some of the latter species, seeds
adhere to the soil surface and germinate during rain dispersal, as in Blepharis spp. (Gutterman et al., 1967; Witztum
et al., 1969). Seeds of certain other species stick to the
soil surface and may germinate in rains the following year
or even after many years. In some cases, seeds stick to
the soil surface by means of a mucilaginous layer, such
as in Artemisia sieberi (Gutterman, 1993), Plantago coronopus, Carrichtera annua, Anastatica hierochuntica
(Gutterman and Shem-Tov, 1996, 1997) and Artemisia
monosperma (Huang and Gutterman, 1998).
Seeds with a mucilaginous layer derived from a thick pellicular coating can attain their maximum water-absorbing
capacity very quickly, for example within 2 h in
Artemisia monosperma (Huang and Gutterman, 1999b;
Huang et al., 2000). The seed, especially the upper parts,
remains hydrated for longer (Huang and Gutterman,
1999b) as indicated in Fig. 1. DNA fragmentation studies
show that this allows time for repair of damaged DNA in
the seed embryo before the seeds re-dry in the morning
sun (Figs 3 and 4). Dew deposition for a few minutes is
all that is needed for extensive repair to take place.
In the central part of the Negev desert, there are approximately 190 nights with each year when dew forms for
2 – 12 h (Evenari et al., 1982). A repair mechanism by
re-wetting from any source has been termed ‘preconditioning’ or ‘priming’ by Heydecker and colleagues (Heydecker
et al., 1973; Heydecker, 1977; Heydecker and Coolbear,
1977). This is known to improve the speed and percentage
of germination when appropriate conditions are present for
actual germination, as shown in Artemisia monosperma
(Huang and Gutterman, 1999b). Such a repair mechanism
is likely to increase the longevity of seeds that are on the
sand surface and exposed to desert nights with dew, as
shown in Artemisia sieberi by Evenari and Gutterman
(1976).
There is considerable published evidence linking DNA
repair or the lack of it with the level of germination
success. For example, a feature associated with loss of
281
viability in dry seed is the increasing loss of integrity of
nuclear
DNA
with
increasing
levels
of
low-molecular-weight oligonucleotide fragments (Cheah
and Osborne, 1978; Elder et al., 1987; Elder and
Osborne, 1993; Leprince et al., 1996). When Secale
cereale (rye) embryos that are non-viable are imbibed for
2.5 h, the extent of fragmentation of DNA shows a significant increase, pointing toward the continuous activity of
endonuclease cleavage in the absence of DNA repair
(Cheah and Osborne, 1978; Elder et al., 1987). Embryos
of imbibed dormant Avena fatua (oat) also show an efficient
repair of radiation-induced lesions in the DNA (Boubriak
et al., 1997), and this repaired DNA remains stable when
embryos are re-dried. For both Secale cereale and Avena
fatua seeds, the failure to restore genetic integrity to the
genome on rehydration of an embryo whose DNA is
damaged by desiccation is likely to be a major factor in
determining desiccation tolerance (Boubriak et al., 1997).
The ability to repair DNA when embryos are rehydrated
is therefore critical to maintaining a functional genome.
In addition, the stability of the DNA on desiccation and
the capacity for repair of DNA on rehydration are considered to be integral components of a total desiccation
tolerance mechanism (Elder et al., 1987; Osborne et al.,
2002).
In the present study, the DNA of Artemisia seeds was
examined following DNA damage via gamma-irradiation.
These seeds were exposed to normal nights in the Negev
desert highlands with dew deposited naturally on the soil
surface. The remarkable stability of the pellicle to enzymatic hydrolysis (Wei and Tu, 1980) and the lack of
evidence that it provides a food source at germination,
suggested that it might play a reliable long-term role in controlling hydration of the embryo and also in fixing
Artemisia seeds to sand particles when the pellicles are
moistened to form mucilage (Gutterman, 2002; Huang
et al., 2004). Genes involved in the synthesis of mucilage
by developing seeds of Arabidopsis have been reported
(Western et al., 2000; Penfield et al., 2001; Haughn and
Chaudhury, 2005). It should be emphasized, however, that
the cells involved in pectin polysaccharide synthesis in
Arabidopsis are not the same as those in Artemisia.
In Arabidopsis, epidermal cells of the outer cell layers of
the seed integument produce mucilage between the
primary wall and the plasma membrane (Western et al.,
2004). In Artemisia, the pectin is secreted outside the pericarp of an achene, and so forms an outer pellicle coat to the
whole seed (Fig. 5).
DNA repair occurring during imbibition of water in other
species is known to remain stable after embryos are again
dehydrated (Elder and Osborne, 1993; Boubriak et al.,
1997). The present experiment shows that without their pellicle covering and resulting mucilage production on
wetting, seeds of A. sphaerocephala were unable to repair
DNA that had been damaged by prior gamma-irradiation.
This effect arises because the mucilaginous layer keeps
the embryo sufficiently hydrated for long enough to allow
DNA repair processes to proceed even though germination
per se is not induced. Seeds of A. ordosica, which colonizes
stable but not shifting dune sand, have only a thin pellicle
282
Huang et al. — Pectin Pellicles, Dew and Seed Embryo DNA Repair
and hold dew for several hours less than those of
A. sphaerocephala. Accordingly, there was far less effective
DNA repair of prior gamma-irradiation damage in
A. ordosica. These findings help to understand the ecological importance of mucilaginous layers when wetted for seed
survival in the desert.
Understanding the unusual pellicles of Artemisia species
and their role in DNA repair may offer new opportunities
for the priming and coating of high-value seeds to protect
them from damage during fast desiccation after aerial seedling treatment and during later hydration and desiccation in
field conditions, especially once the desiccation intolerance
stage has set in. Coating seeds with appropriate hygroscopic
material is therefore suggested for aerial seedlings during
ecological restoration of degraded ecosystems, such as
shifting desert sand dunes.
ACK N OW L E D G E M E N T S
We thank The Royal Society of London and The Chinese
Academy of Sciences for supporting this joint research
project. Z.H. acknowledges the financial support by an
innovative group research grant (30521002) from the
National Natural Science Foundation of China, the Key
Basic Research and Development Plan of China
(2007CB106802), Key Project of CAS (KZCX2-XB2-01)
and National Natural Science Foundation of P.R. China
(30000021, 30570281). We thank Professors Carol and
Jerry Baskin for critical review of this manuscript, and
Dr Heather Davies of the Electron Microscopy Unit, The
Open University, UK, for her valuable advice.
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