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Structural and Functional Differentiation of Bundle Sheath and Mesophyll Cells in the Lamina Joint of Rice Compared with that in the Corresponding Region of the Liguleless Genotype

Abstract The structural and functional characterization of the blade-sheath boundary region of a rice cultivar T65 and its near-isogenic line T65lg were examined by light and electron microscopy and in situ hybridization. Starch accumulation in bundle sheath cells was compared between the lamina joint of T65 and the corresponding region of T65lg and also between the lamina joint and the leaf blade. In the lamina joint of T65, starch grains were predominantly accumulated in bundle sheath cells, and the starch-containing chloroplasts within these cells were spherical in shape. On the other hand, in the blade-sheath transition region of T65lg, little starch accumulation was observed and the chloroplasts were oval in both mesophyll and bundle sheath cells. Furthermore, photosynthesis-related genes, rbcS and cab, were expressed in mesophyll cells within the blade-sheath transition region of T65lg as in the leaf blade and sheath, while no expression of these genes was found within the lamina joint of T65. These facts indicate that T65lg can not develop the lamina joint from either structural or functional aspect. The present results suggest that the control mechanism of starch accumulation in bundle sheath chloroplasts in the lamina joint differs from that in leaf blade in rice.

A rice leaf is composed of sheath, lamina joint and blade, acropetally in the longitudinal axis. The lamina joint is known as gravitropic region, which contributes to bending the blade horizontally from the main axis (Furusawa et al., 1996). In the lamina joint of rice, sclerenchyma is delignifi ed to become collenchyma and vascular bundles are located distant from epidermis (Maeda, 1961), as reported for Panicum maximum and several other species of Poaceae (Paiva and Machado, 2003). Bundle sheath cells of emerging rice leaves predominantly accumulate starch grains but the accumulated starch is dissipated at leaf maturation (Miyake and Maeda, 1976;Tsutsumi et al., 2006). Counterparts of bundle sheath cells in the lamina joint also accumulate large amounts of starch in mature leaves and these starch grains move according to the gravitational vector (Nakano and Maeda, 1978).
In C 4 plant leaves, structural and functional differentiation of bundle sheath and mesophyll cells has been the object of numerous investigations on the regulation of gene expression (Dengler and Nelson, 1999;Sheen, 1999). However, the function of the bundle sheath cells in leaves of C 3 plants is almost unknown (Yoshimura et al., 2004), in spite of the fact that chlorenchyma of leaves in most C 3 plants is also differentiated into bundle sheath and mesophyll cells (Esau, 1953). The bundle sheath of C 3 plant is also intriguing for investigation on the evolution of C 4 photosynthesis and introduction of C 4 genes into C 3 plants (Miyake, 1999). Therefore, the bundle sheath cell-specifi c starch accumulation in the lamina joint of rice, a C 3 plant, is an interesting property to investigate the specifi c role of bundle sheath cells of C 3 plants.
We previously reported that starch was specifi cally accumulated in bundle sheath cells of developing leaf blades and of the lamina joint in mature leaves of rice plants (Tsutsumi et al., 2006). This fact indicates that the bundle sheath cells possess functions different from those of mesophyll cells, at least with respect to sugar transport or starch synthesis (Miyake, 1999). However, it is not yet clarifi ed whether there are functional differences in bundle sheath cells of the lamina joint and the leaf blade.
In addition, we also reported that the expression of photosynthesis-related genes, encoding the s m a l l s u b u n i t o f r i b u l o s e -1 , 5 -b i s p h o s p h a t e carboxylase/oxygenase (rbcS) and the light harvesting chlorophyll a/b binding protein (cab), was suppressed in the lamina joint despite the occurrence of granal chloroplasts, while these genes were expressed in mesophyll cells of mature leaf blades (Tsutsumi et al., 2006). These facts also suggest that the lamina joint is functionally different from the leaf blade.
It is known that there are spontaneous liguleless lines in rice. These liguleless lines are not competent in bending leaf blades, while auxin-treated normal rice leaf blades bend at the lamina joint (Maeda, 1962). This fact indicates that liguleless rice lacks the function of the lamina joint. There are also several liguleless lines in maize. These liguleless maize lines have been studied to investigate the mechanisms regulating leaf development (Sylvester et al., 1996). However, structural and functional differentiation in the inner structure, especially with respect to bundle sheath and mesophyll cells, have not been investigated in detail. Although the lamina joint of rice has specialized structure and function as mentioned above, the bladesheath transition region of liguleless rice has not been studied in detail.
In this paper, we report the structural features and the expression of photosynthetic genes in the region corresponding to the lamina joint of a liguleless line of rice. We discuss the difference in the function of bundle sheath cells between the lamina joint and the leaf blade.

Plant materials
A rice cultivar (Oryza sativa L. cv. Taichung 65) (T65) and its near-isogenic liguleless line T65lg were used. The liguleless gene of T65lg had been introduced from H-79, a linkage tester of Hokkaido University (Oka, 1974), into T65 as a recurrent parent by backcrossing eight times. Seeds of T65 and T65lg were surface sterilized with 5% sodium hypochlorite solution for 10 min. After washing several times with distilled water, seeds were imbibed in distilled water in a growth chamber at 28ºC for 3 days. After imbibition, seeds were sown on plastic nets placed on the surface of 250 mL distilled water in tall beakers. Seedlings were grown in a culture room under continuous light (ca. 75 µmole m -2 s -1 of photosynthetically active radiation (PAR)) at 28ºC for 6 days until the second leaves expanded.

Scanning electron microscopy (SEM)
Segments of 2 mm in length were cut with a razor blade from mature second leaves including the lamina joint in T65 or its corresponding region in T65lg. The samples were fi xed in 5% glutaraldehyde in 0.05 M phosphate buffer (pH 7.2) and then dehydrated with grading ethanol series. For critical point drying, isopentyl acetate was used as intermediate liquid for introducing CO 2 . Critical point drying was carried out in a Hitachi HCP-2 apparatus using CO 2 as the transition. These specimens were coated with platinum with ion sputtering apparatus, Eiko IB-3, and observed under a scanning electron microscope (Hitachi S-4200).

Starch staining
Segments of 5 mm in length were cut with a razor blade from the lamina joint and its corresponding region of the mature second leaves including the emerging third leaves. The samples were fi xed in FAA (70% ethanol: acetate: 37% formaldehyde solution = 18 : 1 : 1) and embedded in paraffi n after ethanol and t-butanol dehydration. Tissue sections (10 µm thick) were mounted on glass slides. Then, starch grains were stained with I-KI solution containing 2.5 g L -1 iodine and 5 g L -1 potassium iodide for 20 min. These sections were observed under a microscope (Olympus BX51).

In situ hybridization
The samples embedded as above were sectioned and mounted on glass slides treated with silane. Then, the sections were hybridized with RNA probes for rbcS and cab prepared as Tsutsumi et al. (2006) and the hybridized probes were detected immunologically with anti-DIG alkaline phosphatase (Roche) and stained with 4-nitroblue tetrazolium chloride and 5-bromo-4chloro-3-indolyl-phosphate. Microscopic observation was conducted as above.

Transmission electron microscopy (TEM)
The method of electron microscopic observation of lamina joint chloroplasts was according to Tsutsumi et al. (2006).

Stability of bundle sheath starch grains
To examine the stability of starch grains in the lamina joint and the emerging leaf blades, we separated the shoots of rice plants grown as above from the seeds and roots and kept them in the dark for 1 to 3 d with the basal 1 cm portion immersed in distilled water. Observation of starch grains was conducted as above.

Surface structure of the lamina joint of T65 and the corresponding region of T65lg
The blade of T65 was inclined at the lamina joint by sensing gravity force (Fig. 1A). The inclination of blade was increased by treatment with 5 mg L -1 IAA ( Fig.  1C; Maeda, 1962). In contrast, the inclination of blade was not induced in T65lg either by gravity force or IAA (Fig. 1B, D).
The leaf of T65 consisted of the blade (Fig. 2B), sheath (Fig. 2E) and the lamina joint (Fig. 2C, D) with the ligule (Fig. 2A). The adaxial surface of leaf blade of T65 showed crenulated cell shape and had a great number of verrucas, occasional hairs and several rows of stomata (Fig. 2B). On the other hand, the cells on the adaxial surface of the leaf sheath were noncrenulated rectangular in shape and possessed no specialized structures on the surface (Fig. 2E), except that stomata were occasionally observed at a much lower frequency than the leaf blade. The adaxial surface of the lamina joint showed cell bulges but no other specialized structures (Fig. 2C, D). The abaxial surface of the lamina joint of T65 was smooth in contrast with that of leaf blade and sheath in which many verrucas were observed (data not shown).
The leaf blade-sheath boundar y of T65lg is indistinguishable when observed macroscopically (Fig. 1). To clarify the blade-sheath boundary, we observed on the surface of T65lg (Fig. 3). The adaxial surfaces of both leaf blade and sheath of T65lg were similar to those of T65 (Figs. 2B, E and 3B, E): The adaxial surface of the leaf blade showed a crenulated cell shape and possessed hairs, stomata and many verrucas, whereas adaxial surface of leaf sheath showed a noncrenulated rectangular cell shape and possessed no specialized structures on the surface. These results indicate that we can distinguish leaf blade from leaf sheath in T65lg based on the adaxial surface structures, especially the verrucas.
In addition, it was evident that T65lg leaf lacked the characteristic lamina joint region ( Fig. 3A compared with Fig. 2A). The blade-sheath transition region of T65lg exhibited transitional feature from blade to sheath (Fig. 3D). Wax and verrucas were reduced basipetally from the leaf blade. The blade-sheath boundary of T65lg was not straight but irregular through the leaf width (Fig. 3A, C), in contrast to the more or less straight boundary between leaf blade and the lamina joint of T65 (data not shown).
There is no obvious difference between the abaxial surface of leaf blade and sheath in either T65 or T65lg. The abaxial surface of leaf blade and sheath was similar to the adaxial surface of leaf blade (data not shown).

Anatomy and cell specifi c starch accumulation in the lamina joint of T65 and the corresponding region of T65lg
The anatomy of the lamina joint of T65 and the corresponding region of T65lg was different. In the lamina joint of T65, several small vascular tissues were observed on the adaxial side near the midvein region of the leaves (Fig. 4C, adV), in addition to the vascular bundles in the abaxial side which were distant from epidermis ( Fig. 4C; Maeda, 1961). On the other hand, vascular tissues in the adaxial side of the corresponding region of T65lg did not develop as in the lamina joint of T65 and the vascular bundles in the abaxial side were not apart from epidermis (Fig. 4G). The lamina joint of T65 was thicker in the transverse section than the corresponding region of T65lg (Fig.  4C, G). In a transverse section of the blade-sheath transition region of T65lg, both the blade-like region with verrucas and the sheath-like region without verrucas were observed (Fig. 4G).
Starch accumulation was observed in bundle sheath cells including bundle sheath extension cells in the lamina joint of T65 (Fig. 4C), the result being consistent with the previous reports (Nakano and Maeda, 1978;Tsutsumi et al., 2006). On the other hand, bundle sheath cells of the blade-sheath transition region of T65lg accumulated few starch grains (Fig. 4G).
Bundle sheath and mesophyll cells of the mature leaf blade of both T65lg and T65 accumulated few starch grains (Fig. 4B, F). The bundle sheath and mesophyll cells of mature leaf sheath of both T65lg and T65 also accumulated few starch grains (Fig. 4D, H). In addition, common adaxial surface characteristics of T65 and T65lg, namely verrucas on the blade and a lack of verrucas on the sheath observed by SEM, were also recognized in longitudinal sections under a light    microscope (Fig. 4A, E). Starch grains in bundle sheath and bundle sheath extension cells in the lamina joint of T65 were more stable than those in emerging third leaf blades when the plants were kept in the dark. The starch grains that had been accumulated in bundle sheath cells of emerging third leaves completely disappeared within 3d of darkness while those of the lamina joint remained (Fig. 5). This indicates that the starch grains in bundle sheath cells of lamina joint are more than reserve material and suggests that a different mechanism is involved in the starch metabolism in bundle sheath cells between the emerging leaf blade and the mature lamina joint.

Expression of photosynthesis-related genes in the mature lamina joint of T65 and the corresponding region of T65lg
We previously reported that the expression of photosynthesis-related genes was not found in mature lamina joint, despite the existence of mature chloroplasts (Tsutsumi et al., 2006). To examine the expression of photosynthesis-related genes in bladesheath transition region of T65lg, we examined the expression of rbcS and cab by in situ hybridization.
The expression of rbcS was observed in mesophyll cells in mature leaf blades of both T65 and T65lg but not in bundle sheath cells (Fig. 6A, D), which coincided with previous reports (Kyozuka et al., 1993;Matsuoka et al., 1994). The rbcS expression in mesophyll cells was also observed in the leaf sheath (Fig. 6C, F). On the other hand, the expression of rbcS was not found in the lamina joint of T65 (Fig. 6B), which coincided with our previous results (Tsutsumi et al., 2006). In the blade-sheath transition region of T65lg, the expression of rbcS was observed only in mesophyll cells (Fig 6E), the result being comparable with that in the blade and sheath (Fig. 6D, F). The region in which the expression of photosynthetic genes was suppressed as in the lamina joint of T65 was not found. This indicates that T65lg could not develop the lamina joint region from the aspect of photosynthetic gene expression.
The expression pattern of cab was almost the same as that of rbcS (data not shown).

Ultrastructure of bundle sheath plastids within the lamina joint of T65 and the corresponding region of T65lg
To examine the structure of photosynthetic and starch accumulating apparatus within the blade-sheath transition regions of T65 and T65lg, we conducted TEM observation (Fig. 7). In the lamina joint of T65, chloroplasts of bundle sheath cells were spherical in shape and possessed large starch grains and several grana (Fig. 7A), showing features of amylochloroplasts in bundle sheath cells of developing leaf blades (Miyake and Maeda, 1976), whereas mesophyll chloroplasts possessed small or no starch grains (Fig. 7B).
In the leaf blade-sheath transition region of T65lg, starch grains in bundle sheath chloroplasts were smaller than those in the lamina joint of T65 (Fig. 7C). This observation corresponds to the results obtained by light microscopy (Fig. 4G). The ultrastructural features of bundle sheath chloroplasts of T65lg are similar to those of the mesophyll chloroplasts (Fig. 7D). The ultrastructure of bundle sheath and mesophyll chloroplasts of T65lg was similar to that of the basal region of the leaf blade just above the lamina joint of T65 (Fig. 7E, F).
The results of the present study are summarized in Table 1.

Ligule of grass species and liguleless mutant
Grass species develop the ligule between the leaf blade and the sheath, although several species such as some species in Echinochloa normally lack the ligule (Chaffey, 2000). Many genes are involved in the ligule development in the grass species. In rice, there are three genes known to affect the development of the ligule: auricleless (aul ), liguleless (lg ) and collarless (col ) (Kurata et al., 2005). The aul mutant does not possess auricles but rudimental ligules. The col mutant lacks collars (Sanchez and Khush, 1998). The lg rice lacks the ligule, auricle and lamina joint (Maekawa, 1988). T65lg used in the present investigation carried lg gene in the genetic background of T65.
In grass species, maize has been the most frequently used for studying the leaf development mechanisms. The morphological events during the development of lamina joint region have been clarifi ed by using a liguleless mutant (Becraft et al., 1990;Moreno et al., 1997). In addition, Sylvester et al. (1990) suggested that the ligule itself is not required to differentiate blade from sheath. On the other hand, the lamina joint region of rice has not been investigated in detail.

Structural features of the blade-sheath transition region of liguleless rice
We observed the adaxial surface of the blade-sheath boundary region of both liguleless and normal rice by SEM. The blade-sheath boundary region of liguleless maize has been observed by SEM in detail during leaf development (Becraft et al., 1990;Harper and Freeling, 1996;Walsh et al., 1998). Our fi ndings obtained by SEM from T65lg are similar to those of liguleless maize in that gradual transition from blade to sheath occurs in longitudinal axis, there is a non-straight blade-sheath boundary and the ligule region consisting of ligule, auricle and lamina joint is absent. These features were confi rmed in both transverse and longitudinal sections by light microscopy (Fig. 4). These facts indicate that T65lg can not develop lamina joint and that the leaf blade gradually transforms to sheath along the longitudinal axis.

Starch accumulation and the expression of photosynthesis-related genes
Large starch grains were accumulated in bundle sheath cells of the lamina joint (Fig. 4C). They were shown to move along the gravity vector and are speculated to function in gravity-sensing (Nakano and Maeda, 1978). In contrast, little starch accumulation was observed in bundle sheath cells of blade-sheath transition region of T65lg (Fig. 4E, G). These small starch grains did not move along the gravity vector (data not shown). These results indicate that bundle sheath specifi c starch accumulation in mature leaves is correlated with the differentiation of lamina joint.
The bundle sheath cells of the emerging leaves accumulate large starch grains (Miyake and Maeda, 1976;Tsutsumi et al., 2006). The starch grains accumulated in bundle sheath cells of the lamina joint are more stable than those of emerging leaf blades under a dark condition (Fig. 5). These facts suggest that starch grains accumulated in bundle sheath (−, not detected; ±, low; +, high; ++, very high) *These starch grains persisted longer under the dark condition than those of the leaf blades. cells in the lamina joint are not for temporal storage as in emerging leaf blades but are the functional component for gravity-sensing.
The expression of photosynthesis-related genes, rbcS and cab, was observed in mesophyll cells in the blade-sheath transition region of T65lg. This feature is similar to that in leaf blades and sheaths of both T65 and T65lg and indicates that the leaf blade is gradually replaced by the sheath along the longitudinal axis in T65lg. On the other hand, no expression of photosynthetic genes was found in either mesophyll or bundle sheath cells of the lamina joint of T65, which corresponds to our previous report (Tsutsumi et al., 2006). The results of starch accumulation and photosynthetic gene expression indicate that T65lg lacks the lamina joint from both structural and functional aspects.
Plastids observed in bundle sheath cells of lamina joint of T65 were spherical in shape, accumulated large starch grains and possessed grana and thylakoids (Fig. 7A). Mesophyll chloroplasts in lamina joint of T65 also possessed developed grana and thylakoids (Fig. 7B). However, photosynthetic genes were not expressed either in bundle sheath or mesophyll cells of lamina joint (Fig. 6B). These results suggest that the lamina joint possessed very low photosynthetic activity if any.

Bundle sheath cells within the lamina joint are functionally different from those in emerging leaf blades
The bundle sheath cells of both lamina joint and emerging leaf blades accumulate large starch grains. The starch grains in bundle sheath cells of leaf blades were accumulated during emerging stage and disappeared in expanded stage, whereas the bundle sheath cells of the lamina joint accumulated starch grains in mature leaves.
The starch grains accumulated in bundle sheath cells of emerging leaves disappeared under the dark condition, while those of mature lamina joint persisted. The expression of photosynthesis-related genes in the lamina joint of T65 was suppressed, whereas that in the blade-sheath boundary region of T65lg was not. The present results suggest that the properties of lamina joint, such as bundle sheathspecifi c starch accumulation and suppression of photosynthetic gene expression, are regulated in the down-stream of Lg gene. The present fi ndings also suggest that starch accumulation in the lamina joint is controlled by mechanisms different from those in the bundle sheath cells of emerging leaf blades.