Origins and Nature of Vessels in Monocotyledons. 13.

Scanning Electron Microscopy

Studies of Xylem in Large Grasses
Author(s): Sherwin Carlquist and Edward L. Schneider
Source: International Journal of Plant Sciences, Vol. 172, No. 3 (March/April 2011), pp.
345-351
Published by: The University of Chicago Press
Stable URL: https://www.jstor.org/stable/10.1086/658155
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Int. J. Plant Sci. 172(3):345–351. 2011.
Ó 2011 by The University of Chicago. All rights reserved.
1058-5893/2011/17203-0004$15.00 DOI: 10.1086/658155

ORIGINS AND NATURE OF VESSELS IN MONOCOTYLEDONS. 13. SCANNING

ELECTRON MICROSCOPY STUDIES OF XYLEM IN LARGE GRASSES
Sherwin Carlquist1 ,* and Edward L. Schneidery
*Santa Barbara Botanic Garden, 1212 Mission Canyon Road, Santa Barbara, California 93105, U.S.A.; and yDepartment of
Horticultural Science, University of Minnesota, St. Paul, Minnesota 55108, U.S.A.

Scanning electron microscopy of thick hand sections of the giant thick-stemmed grasses Arundo donax and
Phyllostachys aurea was used to see how the primary xylem of a monocot with pronounced basal meristem
activity in stems differs from xylem in monocots that show gradual elongation in stems. Several intact
protoxylem vessels with annular thickenings can be simultaneously present in a single bundle, indicating that
redundancy is a possible mechanism for countering loss of integrity resulting from elongation stress. Pro-
toxylem vessels can be large in diameter, even larger than adjacent metaxylem vessels. There is little or no
network of cellulosic fibrils in the primary walls (including end walls before dissolution) of protoxylem vessels,
suggesting that primary walls are essentially gelatinous and therefore may elongate more readily than other
vessel elements studied to date. Annular vessels are much more common than helical ones. Other notable
structural features reported are smoothness of the inside of primary walls in protoxylem vessels, absence of
borders on annuli or gyres of protoxylem vessels (but presence of borders on rims of perforation plates), and
grooves interconnecting one to 10 pit apertures (coalescent pit apertures) on the inside surfaces of metaxylem
vessels. Use of thick sections for study with scanning electron microscopy permits imaging that is ideal for
understanding three-dimensional aspects of xylem anatomy and is recommended over the study of thin
sections.

Keywords: Arundo, bamboo, basal meristem, microstructure, Phyllostachys, protoxylem.

Introduction two genera offered us a chance to determine whether similar

Primary xylem of monocotyledons has been much studied
with the light microscope, and thereby the patterns of second-
ary wall architecture in tracheids and vessel elements are well
known (Cheadle 1942, 1943a, 1943b, 1944; Bierhorst and
Zamora 1965; Cheadle and Kosakai 1971). Xylem of mono-
cots has to date been little studied with SEM. In a recent
study, we looked at primary xylem, especially protoxylem, of
Zingiberales and reported many interesting wall patterns
(Carlquist and Schneider 2010). Zingiberales have regular
and progressive shoot and root elongation. In contrast,
grasses have basal meristem activity, with extensive cell elon-
gation (including that of vascular tissues) at the bases of
nodes, very little at the tops of nodes, and intermediate de-
grees in internode portions. We studied grass stems to com-
pare the xylem structure found in monocots that have basal
meristems with those that do not have basal meristems, such
as Zingiberales. We selected two grasses that have thick
culms and tall canes because they offer abundant vascular tis-
sue and are easily prepared with the techniques we have
adopted. These grasses are Arundo donax L., the giant reed,
and Phyllostachys aurea Riv., yellow bamboo. According to
recent grass phylogeny (Grass Phylogeny Working Group
2001), Phyllostachys is in Bambusoideae, whereas Arundo is
in Arundinarioideae; the widely separated placements of the
1
Author for correspondence; e-mail: s.carlquist@verizon.net.
Manuscript received July 2010; revised manuscript received November 2010.
microstructures can be found in two grasses that are not
closely related. These plants were selected to help us answer
the question of whether functional protoxylem and functional
metaxylem occur concurrently or successively in rapidly elon-
gating grass stems.
Ultrastructural studies of metaxylem vessel development in
angiosperms by TEM reveal a widespread pattern (Esau 1977;
Esau and Charvat 1978; Murmanis 1978; Nidermeyer 1973).
The pit membrane in the end wall of a simple perforation
plate thickens before dissolution. This pit membrane is thicker
than those of the lateral wall pits. The end wall pit mem-
brane shows a three-layered structure—the primary walls of
the two adjacent vessel elements with a middle lamella be-
tween them. As the end wall is hydrolyzed and dissolves, the
pit membranes of the lateral walls become thin and more po-
rous. Relatively few studies of protoxylem have been con-
ducted on an ultrastructural level, but one quite pertinent
to our study is that of Parameswaran and Liese (1978), of
bamboo. That article contains two SEM images, but most of
the photographs were derived from TEM (preparation methods
were not mentioned by the authors).
O’Brien (1974) has related primary wall elongation in pri-
mary xylem tracheary elements to hydrolases liberated dur-
ing autolysis of the protoplast. After hydrolysis, the cellulose
fibrils can stretch passively with cell elongation, unhindered
by the matrix of hemicellulose found in unhydrolyzed pri-
mary walls. These observations confirm what has been seen
with LM (Esau and Hewitt 1940).

345
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346 INTERNATIONAL JOURNAL OF PLANT SCIENCES

Fig. 1 SEM micrographs of vessel elements in xylem from young stems of Arundo donax (A, F) and Phyllostachys aurea (B–E). A, Transection
of protoxylem vessel, showing that the end wall is still intact (the rift is judged to be an artifact); borders are present adjacent to the end wall. B–F,
Longitudinal sections. B, Two annular elements of about equal diameter. A portion of the wall on the upper element was probably stripped away
by sectioning; note the disparity between the two elements in the width of the rings. C, Intact annular element (the rift in the wall was probably
caused by drying) and, below it, two rings from a collapsed element. D, Annular element. The facing part of the wall was stripped away by
sectioning; borders are absent on rings. E, Wide annular element (the rings may have been displaced by the sectioning process) and, above it,
a narrow pitted metaxylem element. F, Two rings (no borders apparent) from an annular element and, below it, a portion of a pitted metaxylem
element.

SEM studies of protoxylem have the unique potential ad-
vantage of being able to demonstrate three-dimensional pat-
terns. Previous SEM studies have used thin sections produced
by microtomes. Such sections, like TEM sections, cannot
access large portions of intact vessel elements in three dimen-
sions, as can SEM. Although SEM does have certain dis-
advantages, we believe that its advantages validate its use in
studies involving both development and microstructure. Thin
sections reveal surfaces for examination that are not greater
or less than the cut surfaces of thick sections. In addition,

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 CARLQUIST & SCHNEIDER—SEM OF XYLEM IN LARGE GRASSES 347

Fig. 2 SEM micrographs of portions of annular vessel elements from longisections of young stems of Phyllostachys aurea (A, B, E, F) and
Arundo donax (C, D). A, Portion of a primary wall, showing pores of various sizes extending between two rings. B, Portion of a ring, with the
facing portion of the primary wall stripped away. No pores are evident in the primary wall. C, Inner face of an element before elongation, with
pores present in the central portion of the primary wall between the rings. D, Inner face of an element before elongation, with irregular breaks
(related to drying) in the primary wall material. E, Strands of wall material connecting a ring to the primary wall of an adjacent cell. F, View of
a primary wall portion between two rings, seen from inside a vessel element. The pores in the wall portion are irregular in size and distribution.

however, thick sections permit greater depth of dimensional-
ity of cells and larger portions of cells than do thin sections.
Therefore, we feel that thick sections have superseded thin
sections in imaging potential. Moreover, thick sections, by
virtue of their stiffness, minimize damage to structures by
handling procedures.
Material and Methods
Actively elongating shoots with internodes of various
lengths were collected in April 2010, after a period of rains
that stimulated new shoots. Arundo donax was collected along
stream banks between More Mesa and the Santa Barbara

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 Fig. 3 SEM micrographs of metaxylem vessels from longitudinal sections of young stems of Arundo donax (A–D) and Phyllostachys aurea (E).
A, Intact end wall separating two vessel elements. A facing portion of the end wall was removed by sectioning; lateral wall pitting shows
characteristic coalescent pit apertures. B, A portion of an intact end wall separating two vessel elements, showing the point of attachment between
the end wall and the secondary wall rim of the perforation plate. The lack of a border may be the result of the filling of such a pit cavity by the

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 CARLQUIST & SCHNEIDER—SEM OF XYLEM IN LARGE GRASSES 349

Municipal Airport in Santa Barbara County, California.
Arundo donax is naturalized along low-lying streams of
southern Santa Barbara County. Phyllostachys aurea was
collected from cultivated material in Hope Ranch, Santa
Barbara County. Stem segments were preserved in 50%
aqueous ethanol. Longitudinal and transverse sections were
prepared with a single-edged razor blade, subjected to three
changes of distilled water, and dried on a warming table be-
tween clean glass slides under pressure. Dried sections were
sputter coated and observed with a Hitachi S2600N SEM. De-
tails of these methods can be found in Carlquist and Schneider
(2006, 2007). The images derived from A. donax do not ap-
pear different from those derived from P. aurea with respect to
any specific characteristics. Because the phenomena repre-
sented are essentially the same in the two genera, the text inte-
grates the genera rather than discussing them separately.
Quantitative data and statistical analysis of these data are
incompatible with the nature of SEM work. Only a limited
number of vessels can be analyzed by SEM examination of
sectioned material. Quantitative data on vessel numbers and
dimensions could be provided by transections and macera-
tions, but even adding those data would not be statistically
sufficient. Quantitative data and their statistical analysis
would be goals of physiological implications, which are not
attempted in our study and require quite different methods.
Results
Protoxylem (Figs. 1, 2)
Perforation plates in grasses are simple and transverse (fig.
1A). The end wall lies between a pair of annuli, which
thereby form borders for the plate. Annuli (rings) of adjacent
protoxylem vessel elements differ markedly with respect to
thickness and diameter. In figure 1B, one sees large, widely
spaced annuli of an earlier-formed primary xylem vessel (top)
as well as narrow, closely spaced annuli of a later-maturing
vessel (bottom). The reverse is true in figure 1C. The inter-
pretation of the relative age of the protoxylem vessels is
based on spacing of the rings.
The primary walls of the vessels shown in figure 1A–1D
are judged to be intact, although artifacts are present because
of preparation: cracks in the end (fig. 1A) and lateral (fig.
1C) walls and vessels cut open (fig. 1B, 1D). The striate ap-
pearance of the outer surface of protoxylem vessels (fig. 1C)
is, at least in part, related to the amorphous material of the
primary wall and their stretching during elongation. The in-
ner surfaces of protoxylem vessels were observed to be
smooth (fig. 1B, lower right; fig. 1D).
As the protoxylem vessels elongate, primary wall breakage
patterns may be seen. The annuli of figure 1E are probably
displaced as a result of sectioning, but thin sheets, porose in
places, interconnect some of the rings. The annuli of figure
1F, further pulled apart, show only shreds of primary wall
material, an appearance also reported by Parameswaran and
Liese (1978) for bamboo protoxylem. Micrographs of the
primary walls of protoxylem vessels at higher magnifications
may show reticulate patterns (fig. 2A, 2F) or expanses of
sheetlike wall material (fig. 2B). At an early stage of develop-
ment, the primary walls of annular vessels (fig. 2C, 2D)
show what appear to us to be accumulations of gel-like wall
material that, when dried, reveals porelike (fig. 2C) or crack-
like (fig. 2D) patterns. As protoxylem elements are pulled
away from adjacent cells, strands of primary wall material
may connect the annuli with the primary walls of adjacent
cells (fig. 2E).
In our work with the microstructure of primary xylem of
various ferns and monocots, we usually see borders on annuli
and on gyres of primary xylem cells. These have been imaged
by investigators using TEM (e.g., Esau 1977). In grasses,
however, annuli lack borders either wholly or mostly (bor-
ders do appear on perforation plates). We have confirmed
this with LM using permanent slides of thin longitudinal sec-
tions of Phyllostachys aurea. In the present study, borderless
annuli may be seen (fig. 1B–1F; fig. 2B, 2D).
Metaxylem (Fig. 3)
Metaxylem vessel elements consist of a large pit traversed
before maturation by a prominent untextured pit membrane
(fig. 3A, 3B, 3D, 3E). The pit membrane of the perforation
plate is apparently thin (fig. 3A, 3B, 3D) compared with the
metaxylem pit membranes viewed with TEM (Parameswaran
and Liese 1978), a difference that is explainable on the basis
of different fixation methods and the tendency of gels to
shrink when dried.
The simple perforation plate of monocots is typically bor-
dered (Bierhorst and Zamora 1965), but borders are not evi-
dent in many of the maturing metaxylem vessel elements of
the grasses we examined (fig. 3A–3D). This may represent
a filling of the border with gels of the primary wall, an ap-
pearance suggested by the smooth flowing contour where the
rim of the perforation plate transitions to the pit membrane
(fig. 3A, 3B). That a border is, in fact, present is suggested
by the perforation plates in which the pit membrane has
been lost by maturation (fig. 3C).
In no immature end wall pit membrane was any porose
or reticulate appearance evident. This was true regardless of
whether the pit membrane was sectioned smoothly (fig. 3A,
3B) or was fractured in some way (fig. 3C, 3D).
The lateral wall pitting of metaxylem vessels of grasses is
more diverse than reports might tend to indicate. Circular
bordered pits are certainly evident on some metaxylem ves-
sels (fig. 3C, 3E) in both of the grasses studied. As seen from
views of the inner surfaces of vessels of Arundo donax, the
occurrence of grooves interconnecting pit apertures is promi-

primary wall gel. C, View of an oblique end wall, seen from outside an element. Borders are present on lateral wall pits, and a border is present on
the perforation; the end wall was stripped away by sectioning. D, Edge-on view of intact end wall (shattered somewhat by the sectioning process),
illustrating the thinness of the dried wall. Extensive grooves are present on the inner surfaces of the two vessel elements. E, Intact primary wall (the
rip in it is an artifact) in face view from an obliquely oriented perforation plate. No fibrillar texturing is visible.

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350 INTERNATIONAL JOURNAL OF PLANT SCIENCES
nent (fig. 3A, 3B, 3D). These grooves (also known as coales-
cent pit apertures) are common in the secondary xylem of
certain dicotyledons (Carlquist 2001). These grooves run in
a transverse or slightly oblique direction (fig. 3A, 3D). The
number of oval pit apertures that may be seen within
a groove ranges from one to 10 or more (fig. 3A).
A peculiar feature seen frequently in our material from
both species is the co-occurrence of relatively wide annual
protoxylem elements and relatively narrow metaxylem tra-
cheary elements (fig. 1E, 1F). All of these are presumably ves-
sel elements, judging from the data of Cheadle (1942).
Discussion and Conclusions
Several facts that have implications for conduction emerge
from our study. Protoxylem vessels are not necessarily nar-
rower than metaxylem vessels. In fact, we observed some no-
tably narrow metaxylem vessels alongside wide protoxylem
vessels. Potentially, this means that large volumes of water per
unit of time could be transported during the earlier stages of
shoot elongation. There is an implication in the plant anatomy
literature, on the basis of the figures one sees (e.g., Esau
1977), that the earliest protoxylem vessels are narrower than
the later-formed protoxylem vessels and that metaxylem ves-
sels are, in turn, larger than protoxylem vessels. One should
not follow such a generalization. In other contexts, one sees
figures of lateral wall pitting of tracheary elements to indicate
the progression from annular through helical to pitted pat-
terns. In these figures, the diameters of all of the tracheary ele-
ments are often, for convenience, shown as identical, whereas
wide variation in the diameters of tracheary elements is the
typical condition. The advantage of thick sections in showing
whole-cell features with SEM is evident from our studies;
hopefully, as thick sections become more widely used, more
accurate representations of tracheary elements will become
more common in textbooks as well as research papers.
Another feature important to the conduction process is that
in several sections we observed an intact, more-elongated pro-
toxylem vessel (annuli widely spaced) next to an intact,
less-elongated protoxylem vessel. Thus, several protoxylem
vessels may be functional concurrently. The methods we have
used show promise for the study of the conductive process in
primary xylem because the status of the primary walls of sev-
eral adjacent vessels can be revealed three dimensionally.
Our images of vessels in the two giant grasses showed that
perforation plates are bordered but that lateral wall annuli
and gyres of primary xylem are not, confirming what can be
seen with LM in longisections of bamboo stems. Typically,
secondary wall thickenings in protoxylem tracheary elements
Literature Cited
Bierhorst DV, PM Zamora 1965 Primary xylem elements and ele-
ment associations of angiosperms. Am J Bot 52:657–710.
Carlquist S 1992 Pit membrane remnants in perforation plates of
primitive dicotyledons and their significance. Am J Bot 79:660–672.
——— 2001 Comparative wood anatomy. Springer, Berlin.
Carlquist S, EL Schneider 2006 Origins and nature of vessels in
monocotyledons. 8. Orchidaceae. Am J Bot 93:963–971.
This content downloaded from 201.141.195.239 on Tue, 11 Sep 2018 02:36:26 UTC
All use subject to https://about.jstor.org/terms
are bordered (Esau 1977), as are the edges of simple perfora-
tion plates in grasses. The extent and functional value of this
feature within grasses need further attention.
The protoxylem of vascular plants that have basal meri-
stem activity in stems, as in the giant grasses, is subject to
rapid elongation. The consequences of this with respect to
microstructure have not been examined in any detail to date.
At least some apparently functional metaxylem vessels occur
along with apparently functional protoxylem in bundles ob-
served for both species. In Zingiberales, which show gradual
basipetal elongation in stems, roots, and inflorescence axes
rather than basal meristem activity, networks of cellulosic fi-
brils may be seen in primary walls of protoxylem (Carlquist
and Schneider 2010). Such fibrils were not observed in pri-
mary walls of protoxylem of the giant grasses. The somewhat
porous primary wall areas occasionally seen in the grasses
can be attributed to shrinkage (drying) patterns of gels rather
than to exposed reticula or meshworks of cellulosic fibrils.
Lack of or paucity of cellulosic fibrils would permit rapid
elongation. We also observed accumulations of what ap-
peared to be gel-like wall material (which contained artifacts
due to drying) between gyres of annular elements that had
not elongated. Although cellulosic fibrils have been observed
in pit membranes of perforations of vessels in certain dicoty-
ledons (Carlquist 1992), no such cellulosic fibril network was
observed in the end wall pit membranes of grass vessel ele-
ments. Much more information is needed to establish whether
cellulosic presence in perforation plates has lessened or disap-
peared phylogenetically.
Another feature that may be related to the rapid elongation
of protoxylem associated with basal meristem activity is the
lack of primary wall patterning in protoxylem vessel elements.
Longitudinal strands of gel-like wall material were observed
in protoxylem vessel primary walls in Zingiberales (Carlquist
and Schneider 2010) but not in the giant grasses. Gel-like
strands that formed attachments between protoxylem vessels
and adjacent cells were, however, observed in the giant grasses.
A feature of special interest that we imaged for Arundo
donax is the abundance of grooves interconnecting pit aper-
tures (coalescent pit apertures). Such grooves are probably
characteristic of many dicotyledons, although reports at
present are relatively few (Carlquist 2001). Although these
grooves can be observed by careful LM work, they may
have been overlooked frequently. Photomicrographs based on
LM are, however, limited in what they can convincingly reveal
about the microstructure of xylem walls. The presence of such
grooves is obvious when SEM is used. The application of SEM
to the study of monocot xylem is relatively recent, and many
more such details are likely to be revealed as more monocots
are studied with SEM.
——— 2007 Tracheary elements in ferns: new techniques, observa-
tions, and concepts. Am Fern J 97:199–211.
——— 2010 Origins and nature of vessels in monocotyledons. 11.
Primary xylem microstructure, with examples from Zingiberales.
Int J Plant Sci 171:258–266.
Cheadle VI 1942 The occurrence of vessels in the various organs of
the plant in the Monocotyledoneae. Am J Bot 29:441–450.
 CARLQUIST & SCHNEIDER—SEM OF XYLEM IN LARGE GRASSES 351

——— 1943a The origin and certain trends of specialization of the
vessel in Monocotyledoneae. Am J Bot 30:1–17.
——— 1943b Vessel specialization in the late metaxylem of the
various organs in the Monocotyledoneae. Am J Bot 30:484–490.
——— 1944 Specialization of vessels within the xylem of each organ
in the Monocotyledoneae. Am J Bot 31:81–92.
Cheadle VI, H Kosakai 1971 Vessels in Liliaceae. Phytomorphology
31:320–333.
Esau K 1977 Anatomy of seed plants. 2nd ed. Wiley, New York.
Esau K, I Charvat 1978 On vessel member differentiation in the bean
(Phaseolus vulgaris L.). Ann Bot 42:665–677.
Esau K, WB Hewitt 1940 Structure of end walls in differentiating
vessels. Hilgardia 13:229–244.
Grass Phylogeny Working Group 2001 Phylogeny and subfamilial
classification of the grasses (Poaceae). Ann Mo Bot Gard 88:373–
457.
Murmanis LL 1978 Breakdown of end walls in differentiating
vessels of secondary xylem in Quercus rubra L. Ann Bot 42:679–
682.
Niedermeyer W 1973 Auflösung der Endwande in differenzierenden
Gefässzellen. Ber Dtsch Bot Ges 86:529–536.
O’Brien TP 1974 Primary vascular tissues. Pages 414–440 in AW
Robards, ed. Dynamic aspects of plant ultrastructure. McGraw-
Hill, London.
Parameswaran N, W Liese 1978 A note on the fine structure of
protoxylem elements in bamboo. IAWA Bull 1978:29–31.

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