PROTEIN STORING VACUOLES IN RAY CEILS OF WILLOW WOOD (SALIX CAPREA L.)
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IAW ABulIetin n. s., Vol. 9 (1), 1988: 59-65
PROTEIN STORING VACUOLES IN RAY CEILS OF WILLOW WOOD
(SALIX CAPREA L.)
by
Jörg J. Sauter and Silvia Wellenkamp
Botany Institute, University of Kiel, Olshausenstrasse 40, D -2300 Kiel, F. R.G.
Summary
Light- and electron-microscopical investi- willow shoots during spring time (Sauter
gations revealed protein bodies of c. 0.5 to 1981). Their total amount reached up to 3.7
2.5 tLm in diameter in the ray cells of willow mg per m1 tracheal sap. But in contrast to
wood. They consist of electron-dense aggre- these weIl known physiological events there
gatesofvarious structural organisation which is still little information on the exact site of
are enclosed in small-sized vacuole-like storage of these nitrogen compounds in the
compartrnents. In semi-thin sections these wood.
aggregates showed positive protein staining Recently, distinct intravacuolar bodies
with Pon~eau Red and Coomassie Blue, and have been described in ray cells of poplar
enzymatic digestibility with pepsin. Because which were suspected to be of proteinaceous
these protein bodies are found during the nature (Sauter & Kloth 1987). Using cyto-
dormant season but not during summer, they chemical protein staining and enzymatic di-
are believed to be specific sites of protein gestion at semi-thin sections further evidence
storage in the ray cells of the wood. This is for their protein nature could be accumulated
in accordance with the biochemical protein (Sauter et al. 1988). Moreover, when the
determination which yielded 6.4 to 8.4 tLg protein extracted from the wood was subject-
mg- 1 dry weight in late fall but only 1.2 to ed to SDS-polyacrylarnide gel electrophore-
2.0 tLg mg-1 dry weight during summer. sis, a peculiar poplypeptide species of an
Key words: Salix caprea L., ray cells, ultra- apparent molecular weight of c. 30 to 32 kilo-
structure, protein storage, protein bodies, dalton was detected. In the present paper
vacuoles, cytochemistry. intravacuolar protein aggregates of similar
appearance are reported for the ray cells of
Introduction the willow wood.
In addition to carbohydrates and lipids, a
fair amount of nitrogen compounds is stored Material and Methods
in the parenchymatous tissues of the wood of The material was collected from c. 7 -year-
trees (e.g., Kramer & Kozlowski 1979). old willow trees (Salix caprea L.) growing in
During the outgrowth of buds in spring, the the Botanic Garden of Kiel University. The
nitrogen content of the wood is known to de- sampies for the microscopical and biochemi-
crease substantially (Tromp 1983). Concom- cal analysis were taken from 2-year-old
itandy, an enormous increase in the content twigs during the winter period (November
of amino acids, amides or other nitrogen until February). For the biochemical analysis
compounds has been observed in the xylem further sampies were collected from May
sap (e.g., Reuter & Wolffgang 1955; Bollard until August.
1960; Tromp & Ovaa 1967) which indicates For the light and electron microscopy ra-
a prominent translocation of such nitrogen dial longitudinal tissue sections were cut
constituents within the xylem itself (see also (thickness c. 250 tLm) using razor blades and
Vogelmann et al. 1985). In addition to gluta- immediately fixed in glutaraldehyde (5%)/
mine, more than 20 different amino acids paraformaldehyde (4%) in cacodylate buffer
have been identified in the tracheal sap of (0.1 M, pR 7.2) for 2 to 4 h at 0 to 2°C. Their
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postfixation with OS04, embedding in Results
Spurr's low-viscosity resin and staining for At the light microscopicallevel, numerous
e1ectron microscopy has been done as de- distinctly stained 'bodies' are detectable in
scribed previously (Sauter & Kloth 1987). all ray cells of the wood during the dormant
The ultra-thin sections were cut with a dia- season (Fig. 1). Following our preliminary
mond knife and viewed in a Siemens 101 results, they have accumulated during fall. In
electron microscope. the procumbent cells of the ray centre, the so-
Semi-thin sections (thickness 0.2 to 0.35 called isolation cells (for terminology see
JlID) of the resin-embedded material were Braun 1970), they appear to be smaller,
used for the light microscopical investiga- reaching c. 0.5 to 1.5 J.l.m in diameter, while
tions. They were stained with or without pre- in the upright cells of the rays they are larger
ceding de-osmification (2% aqueous peri- and may become 2 to 3 J.l.m in diameter (Figs.
odic acid, 30 Min, 21°C) either using Tolui- 1 & 2). Normally these bodies can be hardly
dine Blue (0.05% in 2.5% Na2CÜ3), or, for distinguished from fat droplets or oleosomes.
specific protein staining, with Pon\!eau 2R However, when de-osmification of the resin-
(0.5% in 2% aqueous solution of periodic embedded semi-thin sections is applied be-
acid, pH 1.5; Serva, Heidelberg)or with fore the staining with Toluidine Blue they
Coomassie Blue (0.02% in ethanol-glacial become visible most clearly and can be dis-
acetic acid, 3 : 1). The enzymatic digestion criminated from the oleosomes (see PB and 0
of protein bodies was performed with pepsin in Fig. 2). After this procedure they appear
(pepsin porcine, 1.1 milli ANSON units / mg distinctly blue while the fat droplets stain
from Serva; 0.5% pepsin in 0.1 N HCL with only faintly and assume a yellowish colour in
0.5% Triton X-100; 2 to 5 days at 37°C). In- the microscopic view. The starch grains of
cubation in the same medium but with the the amyloplasts, in contrast, remain white at
pepsin omitted was used as control. the same time (S in Fig. 2). Figure 2 illus-
For the biochemical protein determination trates furthermore the prominent accumula-
tangential longitudinal tissue sections (thick- tion of the three main storage compounds
ness 40 11m) of fresh material were prepared, within the ray cells at the dormant stage, e.g.,
dried in an oven at 80°C (c. 20 h), milled in a the starch, the lipids, and the protein. The
mixer mill (Retsch, Haan, FRG), and ex- starch thereby occupies regularly the centre
tracted with Laemmli buffer (Laemmli 1970). of the cells while the oleosomes and the pro-
An aliquot of the precipitate obtained with tein bodies are typically located towards the
icecold acetone was used for the protein de- cell periphery (Figs. 2 & 3).
termination following the method of Lowry When the de-osmified semi-thin sections
et al. (1951). are stained specifically for proteins with
Figs. 1-4. Ray cells ofthe secondary xylem of 2-year-old twigs of Salb: caprea L. during early
winter stage (December 8, 1986) as seen in radial view with the light microscope (phase con-
trast). Resin-embedded semi-thin sections at x 600 (Fig. 1) or x 1400 magnification (Figs. 2-4).
- 1: Procumbent cells (= IC = isolation cells) and upright cells of the xylem ray showing the
distribution of protein bodies (arrows) within the cells. Toluidine Blue staining after de-osmifi-
cation. N = nucleus; bar = 20 J.l.m. - 2: Ray cells of Figure 1 at higher magnification (x 1400)
showing the accumulation of protein bodies (PB), fat (0 =oleosomes), and starch (S). Note the
larger protein bodies in the upright cells and the occurrence of concave invaginations (arrow-
heads). Toluidine Blue staining after de-osmification. N = nucleus; bar = 10 J.l.m. - 3: Protein
bodies with positive protein staining (Pon\!eau Red staining after de-osmification). N = nucleus,
PB = protein bodies, S = starch; bar = 10 J.l.m. - 4: Dissolution of protein from protein bodies
(arrows) after pepsin digestion (4 days, 37°C). ToIuidine BIue staining. N = nucleus, S = starch;
bar = lOJ.l.m.
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Figs. 5 & 6. Dissolution of protein from a large protein body (PB) after pepsin treatment for 5
days at 37°C (Fig. 6) as compared with the control (Fig. 5). Toluidine Blue staining. Material as
in Figs. 1- 4; x 1600; bar = 10 J.I.I11.
Ponyeau Red (Fig. 3) or Coomassie BIue, the taken from parallel seetions incubated with or
same bodies become positively stained. This without pepsin in the medium.
is taken as an indication of their protein na- At the electron-microscopical level, the
ture. Further evidence for the protein nature same bodies are seen as dark, electron-dense
of these bodies is obtained by incubating aggregates of 0.5 to 2.5 Ilm in diameter
semi-thin sections in a pepsin-containing me- within small vacuoles (Fig. 7). They can be
dium before staining them. After prolonged distinguished clearly from oleosomes that are
incubation of the resin-embedded specimens, stained more homogeneously and are lacking
e.g. up to 5 days, these bodies had almost a delineating membrane (Figs. 7 & 8). At the
completely disappeared (Figs. 4 & 6) while stage investigated, this electron-dense mate-
they remained unaffected in controls lacking rial is filling the vacuoles to a great extent. It
the pepsin (Fig. 5). The pepsin-induced di- shows thereby a granular or reticulate ap-
gestion of a particularly large protein body is pearance. In accordance with the light micro-
demonstrated in Figures 5 and 6 that were scopical pictures this intravacuolar material
Figs. 7 & 8. Procumbent xylem ray cells of Salix caprea L. in the mid-winter stage (February 4,
1987) as seen in radial view with the electron microscope at x 7000 (Fig. 7; bar = 21lm) and at
x 22000 (Fig. 8; bar = 1 Ilm) magnification, respectively. Note the heavy accumulation of elec-
tron-dense material in vacuoles (= protein bodies, PB), of oleosomes (0) and of starch (S) in the
cells. The intravacuolar protein aggregates show often invaginations which occasionally are
occupied by 'multi-tubular membrane complexes' (arrow).
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exhibits often a large concave invagination the contact cell rows at the ray periphery that
(cf. Figs. 2 & 8) which facilitates their iden- correspond with the upright cells in willow.
tification in the light microscope. Because such protein accumulating com-
When the proteins were extracted from partments or vacuoles of seeds are generaIly
the wood with Laemmli buffer and analysed called 'protein bodies' (e.g. Harris & Chris-
quantitatively following the Lowry method, peels 1975; Chrispeels 1983; Saigo et al.
a protein content of 6.4 and 8.4 I!g mg- 1 dry 1983) we apply this term also for the mem-
weight was found in November and Decem- brane-bound protein aggregates in the ray
ber, respectively, while only 1.8 and 1.2 I!g cells of poplar and willow. In earlier studies
mg-1 dry weight were present in May and in of Robards & Kidwai (1969) on the resting
August, respectively. The willow wood thus cambium of Sa/ixjragi/is and of Kidway and
shows a dear-cut seasonal accumulation of Robards (1969) on Fagus sy/vatica, protein
proteins which lies in the range of 4 to 7 I!g bodies also have been reported. However,
mg- 1 dry weight. these bodies differ slightly in structure from
our organelles: they are tightly surrounded by
Discussion an unit membrane and show an amorphous or
In a previous study, a distinct population fine-granular content. Recently, protein bod-
of small-sized vacuoles was found in ray ies of similar appearance to ours have been
cells of poplar in which an electron-dense described at the light-microscopicallevel in
material had accumulated during fall (Sauter bark phloem-parenchyma cells of Sambucus.
& Kloth 1987). Light- and electron-micro- They were shown to contain the Sambucus
scopical investigations revealed that these in- nigra agglutinin (Greenwood et al. 1986).
travacuolar aggregates of c. 0.3 to 1.0 I!m in Details on the origin or on the exact chemi-
diameter consisted primarily of proteins cal composition of the organelles described
(Sauter et al. 1988). This was conduded, in this paper, however, are still unknown.
frrstly, from their staining behaviour when With respect to the frequency and distri-
cytochemical protein staining was used, sec- bution of the protein bodies in the ray cells,
ondly, from their enzymatic digestibility with willow and poplar look very much alike. In
pepsin, and thirdly, from their structural re- poplar between 7 and 13% of the ray cell
semblance with protein bodies described for lumen was found to be occupied by these or-
various storage tissues like cotyledons (Har- ganelles. Thus it is not surprising that the
ris et a/. 1975; Davey & Van Staden 1978; protein content deterrnined biochemically for
Adler & Müntz 1983) and endosperm cells the wood of branches is at fairly the same
(e.g., Saigo et a/. 1983). The present results height in these species. It lies between 5 to 8
confrrm now the occurrence of similar protein I!g mg- 1 dry weight. The great difference ob-
storing vacuoles in ray cells of willow. Their served in the protein content between sum-
size is somewhat larger than in poplar and mer and winter time, e.g., up to 4 to 7 I!g
reaches 2 or even 3 I!m in diameter. Again, mg- 1 dry weight, illustrates on the other hand
positive protein staining and enzymatic di- the importance of the ray tissue in storing
gestibility with pepsin is found in these ag- proteins. Moreover, because at the electron
gregates proving their protein nature. The microscopical level protein bodies were en-
observation of an increased size of protein countered in the present study only during
bodies in the upright ceIls as compared with the dormant season but not during summer
the procumbent cells can be taken thereby as (Sauter, unpublished) they must be suspected
an indication that these cells are not only dif- to be the particular sites of protein storage
fering in their anatomy but also in their phys- within the ray cells.
iology. Interestingly, the same observation
was made in poplar (Sauter et a/. 1988) Acknowledgements
where the isolation cells of the ray centre that The skillful technical assistance of Miss
are believed to be particularly suited for the S. Karg and A.Diercks is gratefully acknowl-
radial translocation (see Sauter 1966; Braun edged. The authors also express their grati-
1970) differed in their protein content from tude to Dr. Barbara van Cleve for valuable
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via free accessSauter & Wellenkamp - Protein storage 65
suggestions and to the Deutsche Forschungs- Laemmli, U. K. 1970. Cleavage of structural
gemeinschaft for financial support. proteins during the assembly of the head of
bacteriophage T4. Nature 227: 680- 685.
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