全 文 ：Received 22 Mar. 2003 Accepted 22 Sept. 2003
Supported by the National Natural Science Foundation of China (30371154).
* Author for correspondence. Tel: +86 (0)25 5427325; Fax: +86 (0)25 5428682; E-mail: .
http://www.chineseplantscience.com
Subcellular Localization of Vegetative Storage Protein of Ginkgo biloba
PENG Fang-Ren*, GUO Juan, WANG Gai-Ping
(College of Resources and Environment, Nanjing Forestry University, Nanjing 210037, China)
Abstract: The ultrastructural characteristics and the subcellular localization of vegetative storage
proteins (VSPs) of Ginkgo biloba L. were systematically studied under the electron microscope. Results
indicated that the VSPs of G. biloba were mostly distributed in the small vacuoles of the phloem
parenchyma cells. The VSPs of phloem parenchyma cells were produced in cytoplasm, then separated by
the inflated cisternae of endoplasmic reticulum (ER), plasmalemma invagination or Golgi body vesicles,
resulting in the formation of vacuole filled with proteins. Three kinds of VSPs were detected: granular,
floccular and massive VSPs, which were distributed in different tissue cells and different cells of the same
tissue or vacuoles of the same cells. VSPs accumulated in autumn and kept at high level throughout the
winter. In the following spring, the bud started growing and VSPs were completely mobilized. With new
shoots growth, VSPs resumed accumulating in late summer and early autumn.
Key words: Ginkgo biloba ; vegetative storage proteins (VSPs); nitrogen storage; ultrastructure
Vegetative storage proteins (VSPs) are the main form of
nitrogen storage in many deciduous trees during winter.
The VSPs usually begin to accumulate in late summer and
early autumn, and are highly abundant throughout the
winter. With new shoots growing in spring, the VSPs are
degraded to amino acid to provide nutrients needed for
shoot growth (Tian et al., 1999; Tan et al., 2000; Peng et al.,
2001). Most of the knowledge on VSPs of woody plants
comes from studies of apple and other fruit trees. In recent
studies, one type of organelle, including rich proteins, which
seems to be a protein body of many dicotyledon’s seeds,
has been found in cortex cell, secondary phloem paren-
chyma cell, xylem ray cell of many deciduous trees and
some evergreen needle trees (Wu and Hao, 1986; Tian et
al., 1999). Using light microscopy of half thin section and
electron microscopy of ultra thin section, studies on the
protein staining behavior and the pepsin digestibility
demonstrated that agglutination with membrane in Populus
and Salix had the characteristics of protein. With SDS-gel
electrophoresis technique, a 32-kD protein was extracted
from extracts of poplar wood (Wu et al., 1997), then this
substance in agglutination of vacuole was signed success-
fully by using immunogold method, and the antibody of
this protein body was gained in white rabbits and was proven
to have the characteristics of protein (Coleman et al., 1992).
In recent years, many studies have been conducted on the
type, localization, function, biosynthesis and degradation
mechanism, genetic expression manipulation of VSPs in the
woody plants (Peng et al., 2001). Ginkgo biloba is a cash-
crop tree because of its high-value of fruits, woods, leaves
and greening park. Although there are some studies on fer-
tilization and nutrient dynamics of G. biloba, few reports are
available on the accumulation and cycling of nutrients in
the inner vegetative tissue of G. biloba. In the present study,
we mainly focused on the morphology, function and sea-
sonal dynamics of the VSPs of G. biloba under ultrastruc-
ture for understanding of their mechanism of formation and
accumulation, and providing with a management-oriented
approach for tree-planting, fertilization and seed collection,
based on the principle of nutrient use-efficient management.
1 Materials and Methods
1.1 Plant materials
Samples were obtained from healthy mature shoots of 15
to 25 cm in diameter at breast height (DBH) of Ginkgo biloba
L., in a wood park of Nanjing Forestry University. The 1- to
3-year-old strong shoots in the middle-bottom of tree crown
were chosen. From January to April in 2001, samples were
collected at the same time of phenology observation. In
dormant and rapid developmental phases, samples were
collected, in January, June, July, August and December,
respectively. But in its strong physiologically metabolism
phase (from active to dormant phase) and budbreak and
new shoots’ growing phase, samples were weekly collected
in March, April, September and October, respectively. In
February, May and November, samples were half-monthly
Acta Botanica Sinica
植 物 学 报 2004, 46 (1): 77-85
Acta Botanica Sinica 植物学报 Vol.46 No.1 200478
collected.
1.2 Samples preparation for light microscopy
Materials were cut into 0.2-0.5 cm of fractions includ-
ing cortex and partial xylem, fixed in 70% FAA, washed and
dehydrated through a graded alcohol series, then put in
activation solution, a compound of curing agent Ⅰ and
Technovit 7100, permeated 5-12 h for preventing the chang-
ing of tissue. Then the enough activation plant materials
were put into embedding media, a compound of active
Technovit 7100 and fixation Ⅱ, after embedding medium
agent. Using the AO circumrotate slicer, sections (5-8 mm
in thickness) were made, then taken by the forceps and put
on the glass slide with water, utterly unfold, dried in oven,
then stained, with 0.025% Coomassle blue to show the gross
proteins, Gimsa to process ordinary observation and
Olympus microscope made in Japan to take photos.
1.3 Samples preparation for electron microscopy
Naught point five cm3 of fractions from 1- to 3-year-old
shoots of G. biloba were quickly put into 4% glutaric
dialdehyde with 0.1 mol/L phosphoric acid buffer, fixed for
24 h at 4 ℃, washed three times (half an hour each) with the
same buffer, then fixed in 1% osinic acid the same buffer
solution for 4-5 h at 4 ℃ or indoor temperature. Owing to
the existence of a stone cells— fibrous ring layer in the
stem cortex of G. biloba, tissue penetration of curing liq-
uids was improved by microwave during fixation process.
After a wash in buffer solution, samples were dehydrated
in an alcohol gradient, passed through propylene oxide,
embedded in Epon812, then sectioned using LKB-V type
ultramicrotome. Sectioned were stained with uranium acetate
and lead citric acid, observed and photographed under trans-
mission election microscope of Calendar H-600 type.
2 Results
2.1 Histochemistry examination of VSPs
The observation of storage tissue of G. biloba’s shoots
after histochemistry staining showed that the VSPs in dif-
ferent cells of the shoots existed significant seasonal
variations.
In mid-July (Fig.1), one to several central vacuoles was
distributed in the cells. Large and round nucleus with one
to two nucleolus suspended in the center of cells. This was
a typical parenchyma cell form. Secondary phloem cells
were orderly arranged. The exobiotic preliminary phloem
cells were deformed and torn up by being squeezed. After
stained by protein specificity dyestuff such as Coomassle
blue, no evident color reaction could be found. This indi-
cated that there was no any protein or that the protein
concentration was low in the phase.
By mid-August, some of the parenchyma cells showed
that their central vacuoles were replaced by several differ-
ent-sized small vacuoles. Most of parenchyma cells still
kept the central vacuole form (Fig.2). At this time, the ellip-
tical or macro-fusiformate nucleus was suspended in the
centers of cells. The massive substance was found on the
edges of central vacuoles and the small vacuoles around
tonoplast intine, but occasionally the massive substance
was also found in the center of vacuoles. Because there
were more proteins in phloem ray cells, the color of stained
cells was darker. The proteins began to accumulate in the
cortex and pith cells. Few proteins could be observed around
the tonoplasts in cambial cells.
From early to late October (Fig.3), a large number of
massive proteins were accumulated in the phloem paren-
chyma cells. The thick protein particles were accumulated
along the tonoplast and a large number of particle sub-
stances were also found in the center of small vacuoles. At
the same time, many proteins accumulated in the cortex
cells, but the protein level was low and the volumes were
very small in the cambium cells.
From mid-November to next early February (Fig.4), the
vacuoles were filled with massive proteins. At this stage,
the number of the storage proteins had hardly any change.
The proteins with different numbers were distributed in all
kinds of parenchyma cells, and protein contents in differ-
ent cells of the same parenchyma are different. Vacuoles of
some cells still existed in the form of the central vacuole.
Central vacuoles of cells were not totally replaced by the
small vacuoles, the protein particles still remained in the
central vacuoles, which were mainly distributed along the
edges of tonoplasts. The dispersal storage proteins in the
vacuoles were also in massive form shown from the vertical
section (Fig.4).
From late February to mid-April, the storage protein
gradually decreased with budbreak and new leaves devel-
opment in spring. The slice on 2nd, in March (Fig.5) showed
that in the parenchyma cells around the cambia of the an-
nual shoots’ barks the proteins had almost disappeared,
but in the cells around phloem fibres many proteins still
existed, and in the parenchyma cells around cambia of the
biannual shoots’ barks a few storage proteins remained.
After leaves developing (early April), the storage proteins
in the parenchyma cells of the annual and biannual shoots’
barks had almost degraded.
At late May (Fig.6), the cross section of G. biloba stem
revealed the disappearance of storage proteins in the pa-
renchyma cells. Numerous small vacuoles became the
central large vacuoles, there were hardly any substances
79PENG Fang-Ren et al.: Subcellular Localization of Vegetative Storage Protein of Ginkgo biloba
that showed positive reaction with protein draining char-
acteristic in the large vacuoles. During this period, the stor-
age proteins had completely degraded or the number was
very little, and the tree was in the phase of vegetation fast
growing.
2.2 Seasonal variation of ultrastructural characteristics
of secondary parenchyma tissue cells
Result from electron microscopy demonstrated that the
ultrastructural characteristics of the secondary phloem pa-
renchyma cells changed with growth rhythm variations of
trees in different phonological periods. These ultrastruc-
tural changes mainly included storage substance and or-
ganelles variations. Forms and contents of storage pro-
teins in parenchyma cells of G. biloba shoots in different
periods were obviously different.
2.2.1 Yellow leaf period to leafless period When leaves
became yellow in late summer and early autumn, phloem
parenchyma cells began to have a few storage substances
in it and the number of the storage substances reached its
maximum in the leafless period. From yellow leaf period to
Figs.1-6. 1. A transverse section of the stem, the typical morphology of the phloem parenchyma cells in summer (Jul. 11), ×100. 2.
A transverse section of the stem, the proteins accumulated in the vacuoles (Aug. 18), ×200. 3. The proteins accumulated in the
parenchyma cell (Oct. 9), ×200. 4. The different kinds of proteins accumulated in the vacuoles (Nov. 17), ×200. 5. The disappearing
of the granule protein in the phloem parenchyma cell near the cambium (wide arrow), there are a lot of proteins in the cells near the phloem
fibre, ×100. 6. The disappearing of the storage protein in the phloem parenchyma cell (May 22), ×100. Abbreviations: Ca, cambium;
Cis, cisternae; Er, endoplasmic reticulum; Gi, Golgi body; M, mitochondrion; N, nucleus; P, plastid; Pc, parenchyma cell; Pf, phloem
fibre; Ph, phloem; Piw, plasmalemma invagination; Pr, protein; Rc, ray cell; S, starch grain; Sc, sieve cell; Stc, stone cell; Tp, tonoplast;
V, vacuole; Va, vacuole; W, cell wall; Xy, xylem.
Acta Botanica Sinica 植物学报 Vol.46 No.1 200480
leafless period, the structure of the parenchyma cells
changed evidently. There were many membrane forms and
structure dispersed in the vacuoles, which gradually bent
and divided the central vacuoles into numerous different
small vacuoles (Fig.7). The proteins of different forms were
distributed in these small vacuoles. In most cells, they were
in granular form and randomly dispersed in the center of
vacuoles or along the edges of tonoplasts (Fig.8). In some
81PENG Fang-Ren et al.: Subcellular Localization of Vegetative Storage Protein of Ginkgo biloba
cells, the proteins uniformly dispersed in the vacuoles in
floccular form (Fig.9). In some other cells, the proteins ad-
hered to the inner tonoplasts in a massive form (Fig.10).
Different forms of proteins were visible in different vacu-
oles in some cells: the protein was in granular form in one
vacuole, but in floccular form and uniformity dispersed in
the other vacuoles (Fig.11). In addition, a few granular or
massive proteins were found in the cytoplasm (Figs.10, 12),
possibly there was no enough time for them to be invested
by membranes. The storage proteins were accumulated in
the phloem from the outer to the inner. When leaves began
to fall, a weak accumulation of storage proteins was ob-
served in cells close to the cambia. However, in leafless
period, important storage proteins could be identified in
the phloem parenchyma and in the cambium cells. When
leaves began turning yellow, parenchyma cells containing
storage proteins exhibited many small vesicles stretched
into the vacuoles and were possibly engulfed by the vacu-
oles (Fig.7). This electron-dense vesicle possibly had rela-
tion with the formation and accumulation of the vacuole’s
proteins. In leafless period, this vesicle structure was rarely
found in the cytoplasm.
From yellow leaf period to leafless period, all the phloem
parenchyma cells contained rich ribosomes, endoplasmic
reticulum, some Golgi bodies and many mitochondria (Figs.
10,12-14). With numerous organelles, the cytoplasms gradu-
ally became richer and thicker. From leaf yellow period to
early leafless period, the cell walls showed ungulate inner
projection. Plasmalemmas also became irregular, invagi-
nated (Figs.10, 15) and produced the vesicle structures, in
which there were fibrous substances pushed to the vacu-
oles (Fig.10). Plasmalemmas invaginations and vesicles
could be observed over leaf-fall period. In the late leafless
period, these structures dispersed. During the leaf-fall
period, many tubular and lamellar endoplasmic reticulum
were accumulated in the cytoplasm around parenchyma
cells, and endoplasmic reticulum cisternae inflated (Figs.
16, 17), in which fibrous substances similar to the protein
fiber in the vacuole could be seen (Fig.16). The ribosomes
adhered to some endoplasmic reticulum (Fig.11). The
mitochondria were very plenty over leafless period. A few
of mitochondria were scattered or some distributed together
in the cytoplasm in the form of round, coryne or dumbbell
and unclear ridges (Figs.12, 13, 15). Golgi bodies appeared
close to the cell walls, around which secretory vesicles
were absent (Fig.12).
2.2.2 Budbreak to shoot growing period In spring, with
budbreak and new shoot growth, all kinds of storage sub-
stances were quickly consumed. Before budbreak, cross
sections of G. biloba’s shoots showed that the VSPs in the
vacuoles of the parenchyma cells had begun degrading.
The granular proteins content in the vacuoles decreased.
The clearance appeared in the middle of the vacuoles (Fig.
19). The volume of the vacuoles was reduced and many
vesicles adhered to the edges of some massive proteins
(Fig.18). Most of the proteins of the vacuoles were mainly
in dispersing granular form and only a few of them was in
the massive form, almost none of them was the uniformity
dispersing flocculent proteins (Figs.18-20). Degradation
of the vacuole’s proteins firstly happened in the cells
nearby the cambia, and then expended to the cells far away
from the cambia. As shown in Fig.21, proteins in the small
vacuoles had almost totally degraded.
In this phenological period, all organelles began
changing. The plasmalemma produced invagination and
formed vesicles towards the inner part of the plasmalemma
(Fig.18). There were a large number of mitochondria with
apparent ridges in the cells. The endoplasmic reticulum in
groups appeared in the cells and were mostly smooth with-
out ribosomes. Few randomly distributed endoplasmics
were scabrous with ribosomes (Fig.22). When vacuoles
began fusing, parts of tonoplasts stretched into the near
vacuoles (Fig.19). Figure 23 shows that two vacuoles closed
to each other, touched tonoplasts became unclear and,
possibly, gradually fused each other. Some vacuoles en-
globed many vesicles and their forms became irregular. In
the ray cells, we found many membraniform residual bodies
(Fig.24). After budbreak, the number of plastids decreased,
their volumes were reduced, and the internal lamellar struc-
ture became small (Fig.25). When new leaves developed
Figs.7-15. 7. A number of vesicles having high electron density invade into vacuole (Sept. 26), × 3 000. 8. Some of the cisternal
endoplasmic reticulum distributing around the vacuoles in which flocculent protein materials begin to accumulate (Sept. 8), ×3 000. 9.
Two kinds of proteins: irregularly-shaped lumps proteins in upper cell and flocculent proteins in the lower cell, ×3 000. 10. The granule
protein and highly developed endoplasmic reticulum and plasmalemma invagination surrounded by numerous cytoplasm. The thick
arrow showed the protein has not been surrounded with membrane (Oct. 9), ×3 000. 11. The two different kinds of proteins in the same
vacuole (granule protein and irregularly-shaped lumps proteins)(Oct. 26), ×4 000. 12. The irregularly-shaped lumps proteins having no
membranes distribute in cytoplasm (Oct. 9), ×3 000. 13. The numerous plastid and mitochondria in the cytoplasm, ×5 000. 14. The
mitochondrion and plastid (Feb.16), ×10 000. 15. The endoplasmic reticulum, mitochondria, ribosomes and plasmalemma invagination
(Nov. 29), ×15 000. The abbreviations are the same as in Figs. 1-6.
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Acta Botanica Sinica 植物学报 Vol.46 No.1 200482
83PENG Fang-Ren et al.: Subcellular Localization of Vegetative Storage Protein of Ginkgo biloba
completely, the electron-dense substances in the vacuoles
almost completely disappeared. Only in some vacuoles, the
rare fibrous substances appeared. The small vacuoles were
gradually replaced by several large vacuoles and the plas-
malemmas tended to become smooth (Figs.25, 26).
2.2.3 Rapid wide expending period During the early
diameter increasing, the parenchyma cells of the second-
ary phloem were typical form of parenchyma cells (Fig.26):
a large nucleus, one to several central vacuoles, the thin-
layer cytoplasm contained endoplasmic reticulum mitochon-
dria and Golgi body. During the rapid diameter increasing
period, the central large vacuoles began to accumulate rare
flocculent substances in some cells (Fig.27). The central
large vacuoles split into numerous small vacuoles and few
proteins appeared in it in some cells. While diameter growth
slow down, the different-sized vacuoles in parenchyma cells
had accumulated a large number of the storage proteins. In
this phase, the organelles in the parenchyma cells did not
significantly vary and the plasmalemma kept smooth (Fig.
27).
3 Discussion
3.1 Localization of VSPs
Observation using light microscopy revealed that in cer-
tain seasons, a large number of proteins were distributed in
cortex, phloem parenchyma cells, wood ray and pith of the
shoots of G. biloba. Moreover, protein content in ray cells
was much higher than that in other parenchyma tissues.
Sauter et al. (1989) found that not only all parenchyma cells
in new shoots, but also those in poplar trunks and roots
passing winter accumulated proteins in poplar trees (Hao
and Wu, 1993). Robert et al. (1990) observed some proteins
in the buds, trunks and roots of poplar trees passing winter
(Coleman et al., 1992). Through electron microscopy
observation, VSPs of G. biloba were synthesized in
cytoplasm, accumulated in vacuoles and distributed in dif-
ferent-sized vacuoles in the parenchyma cells.
3.2 Morphology character of VSPs
The VSPs in woody trees were currently classified into
two groups. One is poplar-type. Temperate zone trees such
as Popular spp. , Salix spp. , Acer spp., Sambucus nigra,
Pinus spp., Picea spp. and Abies balsamea, belong to this
group (Peng et al., 2001). The storage proteins of these
trees in autumn and winter appear in the small vacuoles,
which look like the protein bodies in seeds. However, some
people call them vegetative storage proteins vacuoles
(Greenwood et al., 1990). Now people agree with the latter
because it can more accurately reflect the function, the ori-
gin and the morphology character of this structure. The
second is Hevea-type, which comprises tropical zone trees,
such as Hevea brasiliensis (Hao and Wu, 1993). The three
kinds of Melia trees (Langheinrich and Tischner, 1991)
belong to this type, too. Their storage proteins are accu-
mulated in the central cells and are the special storage pro-
teins of the parenchyma cells. Hao et al. (1986) considered
that the difference in the storage proteins’ types between
temperate and torrid zone trees possibly had relation with
their vegetation development characteristics (Wu and Hao,
1986).
In the present study, the storage proteins of G. biloba
evidently belonged to the poplar-type, suggesting that the
proteins accumulate in the small vacuoles. G. biloba is a
tree species widely distributed in China, from the torrid
zone to the north temperate zone. Our sampling place,
Nanjing, is located in the north edge of the subtropical
zone. From the geography station, it possibly approach the
temperate zone, which has long winter and dry season and
leads trees to dormant state, with physiology adaptation
mechanism resembling that of the temperate zone trees, but
the structures of storage proteins seem to be that of the
temperate zone deciduous broad-leaves trees. It needs to
be further studied if the structures of storage proteins of G.
biloba distributed in the south subtropical zone and the
torrid zone are the same as the results of this paper.
Reported VSPs of trees have many forms. Tian et al.
(2000) studied the VSPs of fifteen temperate zone trees, and
considered that VSPs evidently had three different forms:
floccular, massive and protein-body (Sauter et al., 1989).
Figs.16-27. 16. The cisternal endoplasmic reticulum expended and enclosed the granule protein (Nov. 11), ×15 000. 17. There are
fibril-like materials in the cisternal endoplasmic reticulum (Nov. 29), × 20 000. 18. The depolymerization of storage proteins (thick
arrow) and the plasmalemma invagination (thin arrow) (Mar. 2), ×3 000. 19. The granule proteins are enclosed by the vacuole. Note also
the two vacuoles being fused with each other, × 3 000. 20. The flocculent proteins, × 3 000. 21. The disappearing of the storage
proteins in vacuole after the germination (Mar. 21), ×2 000. 22. The cisternal endoplasmic reticulum and mitochondrion (Mar. 21),
×10 000. 23. The two vacuoles being fused with each other (arrows), there are some vesicles in vacuole (Feb. 16), ×8 000. 24. The
remnant of membrane and flocculent proteins in the radial cells (Mar. 15), ×1 500. 25. The fibril-like materials in big vacuole, plastid and
mitochondrion after the leaves spreading out (Apr. 4), ×4 000. 26. The phloem parenchyma cell after the leaves spreading out (Apr. 17),
×3 000. 27. A little of flocculent protein in vacuole (Jul. 11), ×3 000. The abbreviations are the same as in Figs. 1-6.
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Acta Botanica Sinica 植物学报 Vol.46 No.1 200484
Under the electron microscope, the VSPs of G. biloba’s
shoots also present three forms: floccular, granular and
massive, in which the granular VSPs are the second and the
floccular VSPs are the last. On the other hand, three differ-
ent proteins are distributed in different vacuoles of the same
cell. The granular proteins seem to tend aggregately. This
indicated that the granular proteins finally become the mas-
sive proteins. Meanwhile, we found that proteins in vacu-
oles mainly existed in the floccular or granular form at the
beginning of their production, and scarcely in the massive
form. But later, they were accumulated mainly in the mas-
sive form. In the coming next spring, the massive proteins
firstly diminished, at that time the storage proteins mainly
existed in the granular form. It was likely that during the
process of protein accumulation, the ingredients of pro-
teins had varied. For example, because of agglutinant
increasing, the floccular proteins condensed. However, at
the time of degradation, the condensing ingredients of pro-
teins firstly decomposed and transported.
3.3 VSPs formation and accumulation in phloem paren-
chyma cells
The observation of the phloem parenchyma cells in G.
biloba’s shoots in different phenology periods indicates
that the storage proteins in the vacuoles were mainly syn-
thesized in the cytoplasm. Because of high vacuoles pro-
teins production in cells, there were ribosome and some
ribosome attaching the endoplasmic reticulum in its
cytoplasm. At the same time, the massive and granular pro-
teins showed up in the cytoplasm and their surfaces were
not invested by the tonoplasts. There were ventricular en-
doplasmic reticulum cisternates and Golgi body vesicles
around dispersing proteins in the cytoplasm. This indicated
that the ventricular endoplasmic reticulum cisternae had
possibly encapsulated the proteins in the cytoplasm,
therefore, form the proteins which are invested by single
membrane in the vacuoles, and the Golgi bodies take part in
the composition or transportation of proteins. Apart from
that, the vesicles produced by the plasmalemma invagina-
tion also included the fibrous substances, indicating that
plasmalemmas also took part in the formation of storage
proteins in vacuoles.
Zheng et al. (1990) reported that there are three types of
storage protein accumulation in the vacuoles of soybean:
(1) the proteins gradual deposit on the surfaces of
tonoplasts, along with budding separation of the vacuoles;
(2) condensational ball structure accumulation; (3) uniform
distribution of floccular form. Concerning the accumula-
tion of tree’s VSPs, Wu et al. (1997) found that the accumu-
lation of proteins in Dalbergia odorifera was greatly
different from that in temperate zone trees and seeds: the
storage proteins in Dalbergia odorifera stated in the cen-
tral vacuoles; the accumulation of vacuoles proteins was
that the vesicles, including proteins, gradually compounded
and entered into the central vacuoles (Wu and Hao, 1991).
In this study, G. biloba’s storage proteins synthesized in
the cytoplasm and then accumulated in the vacuoles, fi-
nally existed mainly in the massive form. Some storage pro-
teins were accumulated in the undivided central vacuoles.
When the central vacuoles were split into numerous small
vacuoles, the proteins were also accumulated in the small
vacuoles.
3.4 Seasonal variation of storage proteins in the phloem
parenchyma cells
The xylem tissue of perennial trees and the inner bark
accumulated and stored a large amount of carbohydrates,
nitrogen compounds and fats. In recent years, many stud-
ies have been conducted on the seasonal variation of ev-
ery storage compound in the vegetative storage tissues of
trees, especially in deciduous broad-leaved trees of the
temperate zones (Sauter and van Cleve, 1990; Stepien et
al., 1994). The phloem and xylem parenchyma cells of
shoots as well as the cambium cells of many broad-leaved
trees equally accumulate proteins in the form of protein
body (PB) or storage protein vacuole in autumn to meet
new shoots growth before the photosynthesis recovery in
next spring (van Cleve et al., 1988; Wetzel and Greenwood,
1989). The contents of the proteins are distinctly different
in various trees, but from the second and last 10 d in August,
the proteins began to accumulate in the vacuoles of some
cells, till the later autumn and early winter. After the leaves
had fallen out, a large number of proteins were accumu-
lated in the phloem parenchyma cells, meanwhile, some pro-
teins were also accumulated in the cortex and the wood ray
cells. This state continued until February of the next year.
In early March, the proteins in the phloem parenchyma
cells nearby the cambia firstly began to degrade and slowly
spread to the cortex cells. The storage proteins inside the
barks of annual shoots degraded first, and then the ones
inside the barks of biannual shoots degraded slowly and
were utilized.
References:
Coleman G D, Chen T T, Fuchigami L. 1992. Complementary
DNA cloning of poplar bark storage protein and control of its
expression by photoperiod. Plant Physiol，98:687-693.
Greenwood J S, Demmers C, Wetzel S. 1990. Seasonally depen-
dent formation of protein-storage-vacuoles in the inner bark
tissue of Salix microstachya. Can J Bot, 68:1747-1755.
85PENG Fang-Ren et al.: Subcellular Localization of Vegetative Storage Protein of Ginkgo biloba
Hao B Z, Wu J L. 1993. Vacuole proteins in parenchyma cells of
secondary phloem and xylem of Dalbergia odorifera. Trees,
8:104-109.
Langheinrich U, Tischner R. 1991. Vegetative storage proteins in
poplar. Plant Physiol,97:1017-1025.
O’kennedy B T, Titus J S. 1979. Isolation and mobilization of
storage protein from apple shoot bark. Physiol Plant, 45:
419-424.
Peng F-R, Xu B-S , Guo J. 2001. Research progress on the
vegetative storage proteins in woody plants. Chin Bull Bot ,
18:445-450. (in Chinese with English abstract)
Sauter J J, van Cleve B, Weellenkamp S. 1989. Ultrastructural
and biochemical results on the localization and distribution of
storage protein in poplar tree and in twigs of other three species.
Holzforschung, 43:1-6.
Sauter J J，van Cleve B. 1990. Biochemical immunochemical
and structural studies of protein storage in poplar wood. Planta,
183:92-100.
Stepien V, Sauter J J, Martin F. 1994. Vegetative storage pro-
teins in wood plants. Plant Physiol Biochem, 32:185-192.
Tan H-Y, Wu J-L，Hao B-Z . 2000. The season changes of
ultrastructural of parenchyma cells of secondary phloem of
Dalbergia odorifera. Acta Bot Yunnan , 22:461-466. (in Chi-
nese with English abstract)
Tian W-M , Wu J-L , Hao B-Z . 1999. Cytological study on the
vegetative storage protein in Swietenia macrophylla King. Chin
Trop Crops, 20:25-31. (in Chinese with English abstract)
Tian W-M, Wu J-L ,Hao B-Z . 1999. Cytological and biochemi-
cal identification of major storage proteins in Hevea brasiliensis
seed. Chin Trop Crops , 20:1-7. (in Chinese with English
abstract)
Tian W-M , Wu J-L. 2000. Cytological study on the vegetative
storage protein of 15 species trees. Acta Bot Boreali-
Occidentalia Sin , 20:835-841. (in Chinese with English
abstract)
van Cleve B, Clausen S, Sauter J J. 1988. Immunochemical local-
ization of a storage protein in poplar wood. J Plant Physiol,
133:3146-3153.
Wetzel S, Greenwood J S. 1989. Protein as a potential nitrogen
storage compound in bark and leaves of several softwoods.
Trees，3:149-153.
Wetzel S , Greenwood J S. 1989. Seasonally fluctuating bark
proteins are a potential form of nitrogen storage in three tem-
perate hardwoods. Planta, 178:275-281.
Wu J-L ,Hao B-Z . 1986. The storage proteins cells in the sec-
ondary phloem of Hevea brasiliensis stem. Chin Sci Bull , 31:
221-223. (in Chinese with English abstract)
Wu J L, Hao B Z. 1991. Vacuoles proteins in secondary phloem
parenchyma cells of three Meliaceae species. IAWA Bull , 12:
52-56.
Wu J-L , Hao B-Z , Tan H-Y . 1997. Ultrastructural aspects of
formation and accumulation of vacuole proteins in parenchyma
cells of secondary phloem of Dalbergia odorifera. Acta Bot
Yunnan , 19:381-386. (in Chinese with English abstract)
Zheng Y-Z , He M-Y , Hu A-L , Hao S. 1990. Patterns of the
transition of vacuoles into protein bodies in developing coty-
ledon cells of soybean. Acta Bot Sin , 32:97-102. (in Chinese
with English abstract)
(Managing editor: WANG Wei)