Light and SEM Observation of Opal Phytoliths in the Mulberry Leaf

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Flora 218 (2016) 44–50

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Light and SEM observation of opal phytoliths in the mulberry leaf

a a a a a b

O. Tsutsui , R. Sakamoto , M. Obayashi , S. Yamakawa , T. Handa , D. Nishio-Hamane , b,∗

I. Matsuda

Waseda University Honjo Senior High School, 239-3 Kurisaki, Honjo, Saitama 367-0032, Japan

Institute for Solid State Physics, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8581, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Biogenic mineral deposits 25–100 ␮m in diameter were observed by microscopic examination of the

Received 12 April 2015

leaf of mulberry (Morus alba) ‘Ichinose’. Using chemical mapping with scanning electron microscope,

Received in revised form 21 October 2015

the deposits were determined to be opal phytoliths, composed largely of biogenic silica. Phytoliths var-

Accepted 20 November 2015

ied in diameter and were distributed heterogeneously within a leaf. Many phytoliths were observed in

Edited by Alessio Papini

close proximity to small veins. Phytolith growth in the mulberry leaf proceeds by deposition of silicon

Available online 26 November 2015

compounds transferred via the network of cell walls.

Keywords:

Microscopy BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Opal phytolith Mulberry

Microfossil

1. Introduction toliths in plants remain uncertain, requiring additional research

into individual plant species of interests.

Biogenic minerals are an interesting research focus in botany. Leaves of mulberry (Morus alba L.) ‘Ichinose’ trees are an impor-

An “opal phytolith”, alternatively termed a “plant opal” or a “grass tant food source for silkworms in the sericulture industry. Silica

opal”, is a well-known biogenic siliceous body that has attracted is a feeding stimulant for silkworm larvae (Hamamura, 1959;

interest in various research ﬁelds of biology, archaeology, ecol- Hamamura and Naito, 1961). A small amount of silica is essential in

ogy, and technology (Bauer et al., 2011; Conley, 2002; Exley, 2009; the natural or artiﬁcial food for a silkworm (Iwashina, 2003; Kato,

Fauteux et al., 2005; Hirose, 2013; Katayama et al., 2012; Kondo 1978). Mulberry belongs to the Moraceae family, which is known

et al., 2002; Motomura et al., 2008; Neethirajan et al., 2009; Pearsall, to produce an abundance of phytoliths (Kealhofer and Piperno,

1982; Piperno, 1988, 2006; Rovner, 1971, 1988; Zhang et al., 2013). 1998). Phytoliths in Moraceae have been an important focus of

A phytolith is composed of amorphous hydrated silicon dioxide and phytolith analysis in plant taxonomy and, recently, investigations

results from the biosiliciﬁcation (Bauer et al., 2011; Hodson et al., have been extended especially in tropical regions (Kealhofer and

1997; Lanning and Eleuterius, 1985; Parry and Winslow, 1977; Piperno, 1998; Piperno, 1989, 1991, 2006; Runge, 1999; Wallis,

Piperno, 2006; Sangster and Hodson, 2001; Smithson, 1958). Phy- 2003). Given that phytoliths are a natural reservoir of silica, it is thus

toliths are produced in a variety of higher plant families, such as of interest to examine leaves of mulberry ‘Ichinose’ trees, grown in

Poaceae (Piperno, 2006). Through accumulation of phytolith data the temperate zone, for elucidation of the presence, distribution,

from previous research, the taxonomy of phytoliths has been glob- and formation process of opal phytoliths, In the present research,

ally organized and it is widely accepted that the morphology of we carried out a microscopic investigation on leaves of mulberry

phytoliths is typically family- and genus-speciﬁc (Piperno, 2006). ‘Ichinose’ trees.

The interest in phytoliths has now progressed to their roles and

formation processes in plants (Bauer et al., 2011; Law and Exley,

2011). The universal function and dynamic mechanisms of phy- 2. Materials and methods

2.1. Plant material

Leaves of mulberry (Morus alba) ‘Ichinose’ were collected

∗ ◦

from trees growing wild at Honjo, Saitama, Japan (36 12 N, Corresponding author.

◦

E-mail address: [email protected] (I. Matsuda). 139 10 E). The regional climate is temperate and the average

http://dx.doi.org/10.1016/j.ﬂora.2015.11.006

O. Tsutsui et al. / Flora 218 (2016) 44–50 45

Fig. 1. Leaves of mulberry ‘Ichinose’. Scale bar = 5 cm.

◦

temperature ranges between 5 and 25 C. The harvest period was addition, one of the optical microscopes (VHX-1000) allowed us to

from September to November in 2013 and 2014. In total, 160 leaves directly observe a whole leaf intact for the ﬁve sample leaves.

were sampled from 12 branches of eight trees growing at different

sites. The leaves are palmately lobed or characteristically incised 3. Results

(Fig. 1).

The sampled leaves were immersed in a mixture of water and Fig. 2A shows typical optical-microscopic images of mulberry

ethanol (1:10, v/v), and then rinsed and dried. This procedure ‘Ichinose’ leaves. The biogenic deposits were distributed through-

removed the chlorophyll from the leaf tissues, which assisted with out the probed region. The magniﬁed image in Fig. 2B shows that

observation of the anatomical structure of the leaves. Four of the the biogenic deposits with a size of about 50 ␮m were transpar-

◦

sample leaves were incinerated at 600 C for 4 h to make separation ent and the diameter of the spherical center was about 20 ␮m. To

of the biogenic mineral from a leaf. understand details of the shape, the biogenic deposit was exam-

ined by SEM (Fig. 3). Fig. 3A shows images of the deposits, separated

from the leaves by incineration. “Papilla”-shaped deposits, of about

2.2. ␮

Microscopy 50 m diameter, were identiﬁed and the spherical centers of the

structures were about 20 ␮m in diameter. The same structures were

Observations were carried out with two optical microscopes identiﬁed in the leaves, as shown in Fig. 3B.

(GLB-S600MBIT, Shimadzu; VHX-1000, Keyence) and a scanning To clarify the chemical composition, the biogenic deposits were

electron microscope (SEM; JSM-5600, JEOL) to identify anatomical further examined by SEM-EDX analysis (Goldstein et al., 2003;

features and chemical distribution in the leaf. Observation with a Sakai and Thom, 1979). Fig. 4 shows EDX spectra recorded at ﬁve

SEM is a powerful tool with which to study phytolith morphol- sites: (1) center, (2) near-center, (3) exterior, (4) within a neighbor-

ogy (Brandenberg et al., 1985; Fowke, 1995). A combination of ing cell, and (5) cell wall. Peaks in the EDX spectra correspond to

SEM-energy dispersive X-ray (EDX) microanalysis was performed the intensity of ﬂuorescent X-rays from individual elements. Seven

to allow chemical mapping. An EDX spectrum was recorded from elements, namely carbon (C), oxygen (O), sodium (Na), magnesium

the ∅1 ␮m spot. No pretreatment was necessary for microscopic (Mg), silicon (Si), potassium (K), and calcium (Ca), were identiﬁed.

observations of the whole sampled leaves, but gold coating was Prominent peaks of Si were observed in the spectra of sites 1–3.

needed for observation of the separated minerals. Sampled leaves Oxygen showed the second-largest EDX peak in the spherical cen-

were cut into pieces (typically 5 mm ) for electron microscopy. In ter (sites 1 and 2) and at these sites the O peak intensity was larger

Fig. 2. (A) Optical microscopic image of transparent biogenic minerals in the leaf lamina of mulberry ‘Ichinose’. Scale bar = 100 ␮m. (B) Higher-magniﬁcation image of

transparent biogenic minerals. Scale bar = 50 ␮m.

46 O. Tsutsui et al. / Flora 218 (2016) 44–50

than those of the other sites. These results indicated that the cen-

ter of the “papilla”-shaped structure was composed of silica. At the

exterior (site 3), an EDX peak of C exceeded that of O, but the large

Si peak was still apparent. Comparison of the EDX spectra indi-

cated that the spherical center of the “papilla”-shaped structure

and the exterior components were composed of different ratios of

elements. In cells neighboring the “papilla”-shaped structure, EDX

spectra at sites 4 and 5 showed small peaks of Si and O, but large

C peaks. Amounts of Na and K atoms were indicated to be much

lower than those of Si, C, and O atoms, whereas those of Mg and Ca

were extremely low.

For improved understanding of the spatial distribution of the

individual elements, a set of EDX chemical maps of the observed

SEM region were generated (Fig. 5). As expected, the “papilla”-

shaped structure was perfectly represented in the Si and O maps.

On the basis of the Na and K maps, these alkali metal atoms may

be preferentially distributed at the “papilla”-shaped structure. The

Mg map marginally indicates also preferential distribution of Mg

atoms at the structure. In contrast, the structure appeared black

in the C and Ca maps, indicating that most C and Ca atoms were

located in the surrounding cells. The EDX spectra and chemical

maps indicated that the “papilla”-shaped structure is composed

of silica that may be associated with cations of alkali and alkali

earth metal atoms (sites 1 and 2 in Fig. 4). Thus, one can classify

the biogenic mineral as a “papillae” phytolith, in accordance with

Fig. 3. (A) Scanning electron micrograph of the biogenic minerals, separated from

the International Code for Phytolith Nomenclature (Madella et al.,

leaves of mulberry ‘Ichinose’ by incineration. (B) Scanning electron micrograph of

2005). It is noteworthy that water is known to be a solvent of cations

the biogenic minerals in the leaf captured with electron energy of 15 keV. Scale

␮

bar = 50 m. of alkali and alkali earth metal atoms (Schweitzer and Pesterﬁeld,

2010). Thus, these cations were likely absorbed in a soluble state

by the plant from the groundwater.

We next sought to determine the distribution of the phytoliths

in a mulberry ‘Ichinose’ leaf. Fig. 6 shows examples of microscopic

images of a leaf at three different positions of the lamina: the basal

portion, at the margin near an incision, and at the apex, as indicated

by arrows. The images indicated that the phytolith distribution

was heterogeneous. At the margin and the apex, localized regions

contained many phytoliths and the amount was reduced at the lam-

ina margin. However, in the basal portion, almost no phytoliths

were detected near the midribs. To map the phytolith distribution

over the whole leaf, we chose a leaf that contained phytoliths that

were large enough to observe visually. Fig. 7A and B shows images

of both surfaces of such a sample leaf. The image in Fig. 7A has

been laterally reversed to be comparable with Fig. 7B. Small, white

deposits were densely concentrated in certain regions on the adax-

ial surface (Fig. 7B) and, through comparison with Fig. 7A, it was

evident that these regions corresponded with networks of small

veins. Higher-magniﬁcation images showed the presence of dense

(white) and sparse (dark) areas (Fig. 7C). Furthermore, the dark

region included the leaf margin, which is consistent with optical-

microscopic observation of the leaf (Fig. 6). Optical-microscopic

images revealed that the white regions were rich in large opal

phytoliths (Fig. 8A), whereas the dark regions contained decreased

numbers of small phytoliths (Fig. 8B). Large opal phytoliths were

densely distributed close to small lateral veins (Fig. 8C), as observed

in Fig. 7.

4. Discussion

The transparent biogenic minerals observed in the leaf of mul-

berry ‘Ichinose’ were shown to be composed of silica, as mapped

chemically by SEM. The “papillae” phytoliths observed by SEM were

Fig. 4. Energy-dispersive X-ray spectra of a microscopic region containing the bio-

classiﬁed as amorphous hydrated silica, which is termed an “opal

genic minerals (as shown in Fig. 3B) captured with electron energy of 15 keV.

phytolith” (Katayama et al., 2012; Kondo et al., 2002; Pearsall, 1982;

Individual spectra taken at different sites, indicated in the inset scanning electron

micrograph, are numbered. The peaks corresponding to individual elements are Piperno, 1988; Rovner, 1971, 1988). The SEM-EDX spectra showed

labeled. that the chemical composition differed between the center (sites 1

O. Tsutsui et al. / Flora 218 (2016) 44–50 47

Fig. 5. Topographic image and chemical maps from scanning electron microscopic observation of the biogenic minerals captured with electron energy of 15 keV. Each image

was accumulated with 150 frames. Scale bar = 50 ␮m.

Fig. 6. Optical microscopic images of a mulberry leaf captured at the (A) base, (B) incision, and (C) apex of the lamina. Scale bars = 500 ␮m for magniﬁed images.

and 2 in Fig. 4) and the exterior (site 3 in Fig. 4) in the “papillae” network of cell walls. Thus, it is inferred that Si atoms, transferred

phytolith. The SEM-EDX spectra indicate the amount of individual from the cell wall network, are deposited at the phytolith site. It has

elements at the various sites, but are unable to evaluate accurately been known that the biological silica deposition proceeds with the

the chemical stoichiometry owing to the complicated absorption movement of monosilicic acid molecules, Si(OH)4, in a soluble state

and scattering events for the ﬂuorescent X-ray in a plant. How- from the groundwater to intracellular or systemic compartments

ever, the apparent variation in relative intensity among the element in which it is accumulated for subsequent deposition as amorphous

peaks (Fig. 4) indicated that the chemical composition of the phy- hydrated silica through a possible polymerization of the silicic acid

tolith seemingly differed between the portions. molecules (Bauer et al., 2011; Exley, 2009; Piperno, 2006; Zhang

The Si map in Fig. 5 showed bright contrast associated with the et al., 2013). The present ﬁndings in a mulberry leaf are likely con-

network of plant cell walls. This ﬁnding indicated that the transfer sistent to this biosiliciﬁcation scenario. Since existence of silica in

of Si atoms, the main component of an opal phytolith, occurs via the cell walls has been reported previously (Bauer et al., 2011; Currie

48 O. Tsutsui et al. / Flora 218 (2016) 44–50

Fig. 7. (A, B) Images of the abaxial surface (A) and adaxial surface (B) of a sample leaf. The image in (A) is laterally reversed to be comparable with the image in (B). Scale

bar = 1 cm. (C) Higher-magniﬁcation image from (B). Locations of the two incisions are indicated by arrows. Scale bar = 0.25 cm.

and Perry, 2007; Kim et al., 2002; Piperno, 2006; Sakai and Thom, seemingly belong to the one generated as a siliciﬁed epidermal cell,

1979; Sangster and Hodson, 2001), the transporting silicic acid as discussed above.

molecules in the mulberry leaf seemingly involve in the formation We hypothesized from the images shown in Fig. 8 that the chem-

of phytoliths and also in siliciﬁcation of cell walls. ical species that may be silicic acid molecules were ﬁrst transported

Given that the focus of a SEM is sensitive to the topography of through the midribs of the mulberry leaf. Then, the compounds

the sample surface, the observation of both opal phytoliths and a were transferred to the network of small veins and dispersed in

group of plant cells in Fig. 3 indicates that the phytoliths are likely the lamina and subsequently contributed to production of opal

formed in the epidermis or palisade mesophyll tissue in a leaf. If phytoliths. As shown in Fig. 7, the lateral vein network is highly

formation is assumed to occur in the epidermis, it may be inferred inﬂuenced by the leaf shape and its extension is limited by the

that the “papillae” phytolith observed in the SEM images is a spe- leaf margin. In the incised region of the leaf, the vein network was

cialized epidermal cell, such as a trichome, in the center and the conﬁned to a narrow region between the midrib and the lamina

surrounding basal structures in the exterior portion because simi- margin, which often resulted in a comparatively high density of

lar images of the leaf surfaces are often observed in various plant opal phytoliths.

species (Carpenter, 2006). Through a long controversy, silicon uptake by high plants has

Previous electron microscopic studies have shown that a phy- now been considered to be not completely passive process but it is

tolith in Moraceae species, such as Ficus, Artocarpus, and Streblus, also associated with some biological controls (Law and Exley, 2011;

usually assumes a conical or hat shape (Kealhofer and Piperno, Zhang et al., 2013). At the moment, the present results can simply

1998; Wallis, 2003). In the case of Ficus annulara, siliciﬁed epider- be explained by the passive part, nonselective ﬂow of silicic acid

mal cell phytoliths also have been reported (Kealhofer and Piperno, together with other elements from groundwater through the tran-

1998). On the other hand, it has been observed in Ficus species that spiration stream, followed by formation of solid deposits of silicon

a phytolith can take a form of cystolith that grows out of the cell oxides (Piperno, 2006). The major driving force is considered to be

walls of specialized cells, which occur in the epidermis and ground transpiration or water loss. In other words, in actively transpiring

tissue of leaves (Runge, 1999). While a number of phytolith shapes cells, a supersaturated solution of silicon compounds is thought

are reported for Moraceae species, the phytoliths in a mulberry leaf to develop, leading to the precipitation of silica and water loss is

O. Tsutsui et al. / Flora 218 (2016) 44–50 49

Fig. 8. Optical microscopic images of a mulberry leaf shown in Fig. 7 in (A) a region of high phytolith density, (B) a region of low phytolith density, and (C) a vein. Scale

bar = 200 ␮m.

highest in the uppermost leaf. This argument is consistent with the Acknowledgments

fact that phytoliths in a mulberry leaf were observed only on the

adaxial side, which faces the sun (Fig. 7). The further biochemical This work was supported as part of the Super Science High

investigation may reveal that the phytolith growth site is related School program of the Ministry of Education, Culture, Sports, Sci-

to a certain biomolecule (Law and Exley, 2011). ence and Technology of Japan and partly performed using a facility

The microscopic observations described herein, together with at the Synchrotron Radiation Research Organization, the Univer-

previous ﬁndings (Bauer et al., 2011; Exley, 2009; Piperno, 2006), sity of Tokyo. The authors acknowledge T. Kanai, H. Arai, K. Yano,

suggest the following scenario of phytolith growth in mulberry K. Suminokura, and H. Matsushita for assisting with polarizing

leaves. Molecules of monosilicic acid at the lamina base are trans- microscopy. The authors thank M. Hattori and K. Hasegawa for

ported in the midribs and then in the small vein network. The silicic initiating the research and M. Mochizuki for revising the English

acid molecules are then dispersed from the veins and pass through in the manuscript. The authors appreciate Kaori Takeuchi for her

the cell wall network. At the site of phytolith formation, silicic acid experimental support for the SEM observation.

molecules are deposited, possibly through the polymerization, and

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