Introduction

The vascular system in higher plants, which consists of the xylem and the phloem, is distributed throughout the entire body of the plant. The xylem is responsible for the long-distance transport of water and dissolved mineral nutrients, while the phloem is responsible for the transport of the photoassimilates from the leaf to the rest of the plant. The vascular system is also important because it provides the internal skeletal structure to reinforce the mechanical strength of the plant. The study of vascular pattern formation is, therefore, an attractive theme in plant science. Genetic and molecular biological analyses with a model plant, Arabidopsis, have revealed various factors that are involved in vascular pattern formation in leaves. Detailed analysis of mutants defective in vein continuity, such as van3/scr (Koizumi et al. 2005; Sieburth et al. 2006), cvp1 (Carland et al. 2002), and cvp2 (Carland and Nelson 2004), revealed the involvement of ARF-GAP-mediated vesicle transport, sterols and inositol triphosphate signaling pathways. Furthermore, localization of PIN proteins as auxin efflux carriers determines the route of auxin flow, which, in turn, results in the vascular pattern along the route (Nelson and Dengler 1997; Berleth et al. 2000; Sachs 2000; Aloni 2001; Dengler 2001; Turner and Sieburth 2002; Mattsson et al. 2003; Scarpella et al. 2006).

Not much is known about the molecular mechanism of vein pattern formation in monocotyledons, although detailed observations of leaf development in many monocotyledon species have been recorded for maize (Sharman 1942; Esau 1943; Kumazawa 1961; Russell and Evert 1985; Langdale et al. 1989; Bosabalidis et al. 1994), wheat (Sharman and Hitch 1967; Blackman 1971; Patrick 1972), barley (Klaus 1966; Dannenhoffer et al. 1990; Dannenhoffer and Evert 1994), rice (Kaufman 1959; Inosaka 1962; Chonan et al. 1974, 1984), sugarcane (Colbert and Evert 1982), Arundinella hirta (Dengler et al. 1996, 1997), and several species including C3 and C4 grass (Crookston and Moss 1974; Brown 1975; Dengler et al. 1985, 1986; Ueno et al. 2006). Monocotyledons generally have narrow, long, blade-shaped leaves. The cell division in the basal zone of the leaf contributes to the one-directional elongation of the leaf. Depending on the leaf structure, the venation pattern in monocotyledonous plants is simple: longitudinal veins lie along the proximodistal axis, and transverse commissural veins connect adjacent longitudinal veins along the mediolateral axis. This simple vascular pattern enables us to investigate the temporal and spatial regulation of the differentiation processes of distinctive vascular cells during vein formation.

Oriza sativa (rice) is an excellent model plant of monocotyledons. Previous anatomical studies have revealed various aspects of vascular system formation such as venation patterns, including the continuation of the vasculature between leaves and stems, and organized patterns of specific cells in the vascular system in rice plants (Yamazaki 1961; Inosaka 1962; Chonan et al. 1974). Although rice is a very useful material for genetic studies, only a few rice mutants have been examined for their venation patterns (Scarpella et al. 2003). Consequently, research carried out to date on vascular tissue in rice has not revealed how specific vascular cells interact with each other and differentiate to form an organized vascular system.

The objective of this study was, therefore, to determine sequential programs of differentiation into various different vascular cells by detailed observations of the process of vascular development, using serial sections of the base of developing rice leaves where vascular tissue was just beginning to differentiate.

Materials and methods

Plants and growth conditions

Oryza sativa L. Japonica cultivar ‘Taichung 65’ and Zea mays L. cultivar ‘Honey bantam’ were used. The rice seeds were surface sterilized (Rueb et al. 1994) and germinated in the dark at 30°C for 2 days on half-strength Murashige and Skoog (MS) medium in which MS vitamins had been replaced with B5 vitamins and supplemented with 60 g/l sucrose, 0.5 g/l 2-morpholinoethanesulfonic acid (MES) monohydrate, and 8.0 g/l agarose. The germinated seeds were grown in a 14-h light:10-h dark cycle at 30°C. The maize seeds were germinated in soil and grown in a 14-h light:10-h dark cycle at 30°C.

Histology

For serial section analysis of the developmental process of the vascular bundle, a 12 mm-long basal part, including the shoot meristem, was collected from 12 rice seedlings 12 days after germination, and a 15 mm-long basal part, including the shoot meristem, was corrected from 12 maize seedlings 14 days after germination. For observation of mature vascular bundles in leaf blades, 8-mm-long tenth leaf blades and seventh leaf blades were prepared from 20 rice plants and 20 maize plants, respectively. These fresh plant tissues were fixed with FAA solution (5% formaldehyde, 5% acetic acid, 90% ethanol; volume ratios) for 2 days at 4°C. The fixed samples were rinsed twice with phosphate buffer (pH 7.2), dehydrated in a graded ethanol series (50%, 70%, 90%, 99.5% and 100%) for 12 h at each ethanol concentration and then in 100% ethanol for 24 h. The samples were then embedded in Technovit 7100 resin (Kulzer and Co., Wehrheim, Germany), according to the manufacturer’s instructions. Sections (2 μm thick) were cut with an ultramicrotome (Leica Microsystems RM2165, Wetzlar, Germany), dried at 45°C, and stained with 0.01% o-toluidine blue for 2 min in 50 mM citrate buffer (pH 4.4) at 60°C. After being washed with distilled water three times, the samples were mounted in epoxy resin. The mounted samples were observed under an epifluorescence microscope (Olympus BX51, Tokyo, Japan) equipped with a 3 charge-coupled device (CCD) digital photographic camera (Hamamatsu Photonics C7780, Hamamatsu, Japan). The images were processed with Adobe Photoshop CS3. Fixed leaf tissues were also directly observed without sectioning, in accordance with the method of Scarpella et al. (2003) for rice and Kuwabara and Nagata (2006) for maize.

Results and discussion

Developmental processes of large vascular bundles in rice and maize

The basic venation pattern in the monocotyledon is generally very simple: it is composed of longitudinal veins (the parallel veins) running parallel along the proximodistal axis in the leaf, and commissural veins (the transverse veins) arranged at almost the same intervals, like ladders, between longitudinal veins (Figs. 1a, 2a). This pattern, which is widely observed in monocotyledons, is generally referred to as ‘striated’. There are three kinds of longitudinal veins in rice leaves: the midvein, the large vascular bundle (Fig. 1b), and the small vascular bundle (Fig. 1c). On the other hand, there are four kinds of longitudinal veins in maize: the midvein, the large vascular bundle (Fig. 2b), the intermediate vascular bundle, and the small vascular bundle (Fig. 2c). The intermediate vascular bundles extend throughout, from the leaf blade to the leaf sheath, while the small vascular bundles stop at the end of the leaf blade. Because, except for this extension, the developmental processes of the intermediate and small vascular bundles are quite similar, and because it is difficult to distinguish between the intermediate and the small vascular bundles from cross-sections, we generally used the small vascular bundles as the term showing veins formed between the large vascular bundles in maize. The midvein, the largest vascular bundle, is positioned in the middle of the leaf, and several large vascular bundles are positioned in a row, from the central zone toward both sides. In the leaf sheath, each small vascular bundle is arranged between two large vascular bundles (Fig. 1d). In the leaf blade, the number of small vascular bundles between the two large vascular bundles changes in proportion to the width of the leaf (Fig. 1e).

Fig. 1
figure 1

Venation pattern and vascular bundle structure of the leaf blade in rice. a Venation pattern in the tenth leaf blade. Longitudinal veins, large vascular bundles and small vascular bundles run along the proximodistal axis in the leaf. Commissural veins are located between two adjacent longitudinal veins. b A large vascular bundle in the tenth leaf blade having a protoxylem vessel and phloem tissue on the adaxial and abaxial sides, respectively, two distinctive metaxylem vessels, a mestome sheath and vascular bundle sheath. c A small vascular bundle in the tenth leaf blade lacking distinct large metaxylem vessels. d A transverse section of the basal area in a seedling 12 days after germination. Note that each small vascular bundle is positioned between two large vascular bundles in the third leaf sheath. e Magnification of the box in (d). Note that a few small vascular bundles are positioned between two large vascular bundles in the fourth leaf blade. LV large vascular bundle, SV small vascular bundle; CV commissural vein, PX protoxylem vessel; Ph phloem tissue, MS mestome sheath, VBS vascular bundle sheath, Xy xylem vessel. Scale bars a = 250 μm; b–e = 100 μm

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Fig. 2
figure 2

Venation pattern and vascular bundle structure of the leaf blade in maize. a Venation pattern in the fourth leaf blade. b Structure of a large vascular bundle in the fourth leaf blade. c Structure of a small vascular bundle in the fourth leaf blade. Note that these vascular bundles are enclosed by a layer of cells, named the vascular bundle sheath, while those of rice are enclosed by two layers of cells: the mestome sheath and the vascular bundle sheath. LV large vascular bundle, SV small vascular bundle, CV commissural vein, PX protoxylem vessel, Ph phloem tissue, VBS vascular bundle sheath, Xy xylem vessel. Scale bars = 50 μm

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In the vascular bundle of rice plants, the xylem vessels and phloem tissue are arranged in the adaxial and abaxial sides, respectively (Fig. 1b, c). The large vascular bundles in the leaf blade have three xylem vessels: a protoxylem vessel and two large metaxylem vessels (Fig. 1b). The small vascular bundles in the leaf blade have some small xylem vessels (Fig. 1c). These vascular tissues are surrounded by two layers of the mestome sheath and the vascular bundle sheath.

In order to elucidate developmental process of the vascular bundle, we observed serial sections of the basal part of 12-day-old rice seedlings (Fig. 3) and 14-day-old maize seedlings (Fig. 4). To ensure the reproducibility, we examined samples from 12 different seedlings. Observations were carried out from the tip to the base of developing leaves so that we could trace back the cell lineage of the veins. The serial sections were sliced (2 μm thick) from the shoot apical meristem (SAM) up to 5 mm (rice plant) and 8 mm (maize). Developing large vascular bundles in the fourth and fifth leaves were clearly observed. This was because their structures were large enough for us to observe the differentiation of each vascular cell morphologically.

Fig. 3
figure 3

Developmental processes of the large vascular bundle (LV) in rice. Serial transverse sections were obtained from the basal area of a 12-day-old rice seedling. a Overview of a section containing four leaves, from the third to the sixth. Arrows indicate procambium of the LV in the developing fifth leaf blade. b–g Developing LVs in the fourth (c–f) and the fifth (b) leaf blades. b Stage I. The procambium emerges from the middle layer of the ground meristem and forms the circular layer in its outermost zone, which later differentiates to form the mestome sheath. c Stage II. A primary protoxylem vessel and a few phloem cells emerge on the adaxial and abaxial sides, respectively. The development of phloem tissue continues to stage V. d Stage III. A cell adjacent to the primary protoxylem vessel starts expanding and differentiates into a secondary protoxylem vessel. e Stage IV. Two cells adjacent to the circular layer start differentiating into metaxylem vessels. f Stage V. Ground meristem cells surrounding the circular layer start expanding remarkably and differentiating into vascular bundle sheath cells. g Stage VI. The former position of the protoxylem vessel is occupied by the protoxylem lacuna, and the circular layer is differentiated into the mestome sheath. Vascular cell differentiation is completed. h A model illustrating the differentiation process of the LV in the leaf blade of rice. PC procambium, CL circular layer, PX protoxylem vessel, Ph phloem cells, MX metaxylem vessel, VBS vascular bundle sheath, PL protoxylem lacuna, MS mestome sheath. Scale bars a = 250 μm; b–f = 20 μm g = 50 μm

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Fig. 4
figure 4

Developmental processes of the large vascular bundle (LV) in maize. Serial transverse sections were obtained from the basal area of a 14-day-old maize seedling. a–e A LV in the fourth leaf blade. a Stage I. The procambium has emerged from the middle layer of the ground meristem and forms a circular layer in its outermost zone, which differentiates to form the vascular bundle sheath later. b Stage II. A primary protoxylem vessel and a few phloem cells emerge on the adaxial side and abaxial side, respectively. The development of phloem tissue continues to stage IV. c Stage III. A cell adjacent to the primary protoxylem vessel starts expanding and differentiates into a secondary protoxylem vessel. d Stage IV. Two cells adjacent to the mestome sheath start differentiating into metaxylem vessels. e Stage V. The circular layer cells start expanding and differentiating into vascular bundle sheath cells, and the former position of the protoxylem vessel is occupied by the protoxylem lacuna. f A model illustrating the differentiation process of the LV bundle in the leaf blade of maize. PC procambium, CL circular layer, PX protoxylem vessel, Ph phloem tissue, MX metaxylem vessel, VBS vascular bundle sheath. Scale bars a–e = 50 μm

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Repeated observations revealed a conserved developmental process of the vascular bundle in rice plants (Fig. 3). The leaf primordium consists of a layer of epidermal cells and three layers of ground meristem cells. The procambium of the midvein, the first large vascular bundle, was initiated at the center of the leaf primordium at the collar stage, and subsequent procambium of the large vascular bundles was formed along the mediolateral axis (Fig. 3a). The differentiation process of the vascular cells could be divided into six stages. In the earliest leaf stage, the procambial cells started to differentiate in the middle layer of the three ground meristem cell layers. The outermost cells of the procambium formed the circular layer (stage I, Fig. 3b), and cell division in this layer occurred along the radial axis to keep a single cell layer. The circular layer finally differentiated into the mestome sheath but not a vascular bundle sheath. Subsequently, a primary protoxylem vessel and phloem cells differentiated at the adaxial and abaxial sides, respectively, in contact with the circular layer (stage II, Fig. 3c). Phloem development continued even in later stages. After the primary protoxylem vessel had differentiated, one cell adjacent to the protoxylem vessel started to expand, and it differentiated into a secondary protoxylem vessel (stage III, Fig. 3d), and then two metaxylem vessels, which adjoined the circular layer, started to differentiate (stage IV, Fig. 3e). After the metaxylem development was almost completed, the ground meristem cells immediately surrounding the circular layer started expanding remarkably and differentiating into the vascular bundle sheath (Stage V, Fig. 3f). Finally, the protoxylem vessels collapsed, the position was occupied by the protoxylem lacuna, and differentiation of the large vascular bundle was completed (stage VI, Fig. 3g). Usually, between stages II and IV, procambial cells proliferated in the middle cell layer, resulting in an increase in the size of the large vascular bundles (Fig. 3c–e). A model of the differentiation process in the large vascular bundle in rice is shown in Fig. 3h.

The developmental process of the large vascular bundle in maize was almost the same as that of rice until stage IV (Fig. 4a–d), as illustrated in a model shown in Fig. 4f. In stage V, however, the circular layer in maize started to expand, and it differentiated into the vascular bundle sheath without forming any other sheath structure (Fig. 4e), whereas, in rice, the vascular bundle sheath was differentiated from the ground meristem cells and enclosed the circular layer (Fig. 3f, g). These observations suggest that the vascular bundle sheaths in rice and maize are derived from different cell lineages. Langdale et al. (1989) observed the cell lineage of the vascular bundle sheath in maize and proposed two possibilities, namely that vascular bundle sheath cells arose from procambial cells or from surrounding ground meristem cells. The results that we obtained from our observations of the developmental process of maize vascular bundles, and upon comparing them with our observations made for rice, strongly suggest that the vascular bundle sheath cells in maize arise from the procambium.

Developmental processes of small vascular bundles in rice plants

Observations of serial sections in the fourth and fifth leaf blades revealed that the developmental process of small and large vascular bundles is fundamentally the same, except for the remarkable expansion of metaxylem vessels that was observed in the large vascular bundle (Fig. 5a–f). In small vascular bundles of rice leaves, at first, the outermost cells of the procambium formed the circular layer, which eventually differentiated into the mestome sheath in a later stage (Fig. 5a). Then, the procambial cells proliferated to enlarge the size of the procambium, and simultaneously some of ground meristem cells surrounding the circular layer divided radially (Fig. 5b). The number of procambium cells continued to increase but without a significant increase in the procambium size (Fig. 5c), and then some of their descendants differentiated into a xylem vessel and phloem cells (Fig. 5d). Ground meristem cells surrounding the procambium turned into vascular bundle sheath cells (Fig. 5e). In the later stages, the small xylem vessels differentiated, instead of the two large metaxylem vessels observed in the large vascular bundle (Fig. 5e, f). There was no significant difference in structure of the small vascular bundle between rice and maize except for the presence of a mestome sheath layer in rice, which was similar to the case in the large vascular bundle (data not shown).

Fig. 5
figure 5

Developmental processes of the small vascular bundle (SV) in rice. Serial transverse sections were obtained from the basal area of a 12-day-old rice seedling. a–f A SV in the fourth leaf blade. a The procambium of the SV forms a circular layer in its outermost zone, which differentiates into the mestome sheath later. b The procambium of the SV expands in size by cell division. Ground meristem cells surrounding the circular layer show cell division, according to the procambium expansion (arrow). c The number of cells in the procambium of the SV continues to increase, but the size of the procambium does not increase significantly. d A primary protoxylem vessel and a few phloem cells emerge on the adaxial and abaxial sides, respectively. e Ground meristem cells (circled by dotted lines) surrounding the circular layer start differentiating into vascular bundle sheath cells. f Vascular cell differentiation is completed. PC procambium, CL circular layer, Xy xylem vessel, Ph phloem tissue, VBS vascular bundle sheath, MS mestome sheath. Scale bars = 20 μm

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Development of commissural veins in rice and maize

Among the most characteristic tissues in the vascular system in monocotyledons are the commissural veins. Commissural veins are arranged at ordered intervals between two longitudinal veins that run abreast, connecting them. The transverse sectioning of the commisural vein revealed that a commissural vein in the leaf blade of rice contains a xylem cell, a phloem cell and two companion cells (Fig. 6a), which is consistent with the result obtained from the observation of longitudinal sections by Chonan et al. (1974). In order to elucidate when and where the commissural vein starts to differentiate and how it develops, we observed serial sections of vascular tissues in the base of the leaf where the commissural veins were in the process of differentiating. We mainly used rice plants because the intervals between commissural veins in rice are much narrower than those in maize, which allowed us to observe all the stages of vein cell development with serial sections.

Fig. 6
figure 6

Initiation of commissural vein formation. a–c rice; d maize. a A developed commissural vein in the fifth leaf blade. Note that a commissural vein is composed of four cells (asterisks): a xylem vessel, two companion cells, and a phloem cell. b A differentiating commissural vein from a large vascular bundle to a small vascular bundle in the fourth leaf blade. Note that the differentiation of a commissural vein commences at a circular layer cell that is adjacent to both a xylem vessel and a few phloem cells, and it appears to progress in the middle ground meristem in one direction toward the next vascular bundle. Arrows show the periclinal cell divisions in a developing commissural vein. c A differentiating commissural vein between two neighboring small vascular bundles in which only the central cell has not divided. Arrows show periclinal cell divisions. d The initiation of the commissural vein between two neighboring small vascular bundles in maize. Note that the circular layer cell that is adjacent to both a xylem cell and a few phloem cells divides and finally differentiates into phloem and xylem cells, just as in rice. Asterisks show four cells comprising the commissural vein. MX metaxylem vessel, Ph phloem tissue, CL circular layer. Scale bars a–d = 25 μm

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Differentiating commissural veins were detected only in stage V (Fig. 6b, c). In this stage, as mentioned above, metaxylem vessels in large vascular bundles or xylem vessels in small vascular bundles have completed differentiation. The initiation of the commissural vein arose in a circular layer cell that was in contact with both a metaxylem vessel and one or a few phloem cells, in both large and small vascular bundles. In accordance with a previous report (Kaneko et al. 1980), small vascular bundles in fourth and fifth leaf blades of rice seedlings lacked a mestome sheath layer but instead contained larger thick-walled parenchyma cells, which had the same cell lineage as the mestome sheath cells had. The visible sign of differentiation into commissural veins was the periclinal division of the cells (Fig. 6b), which was observed only in the middle layer of the ground meristem. Judging from the observation of synchronous periclinal division, we found that, in most cases, a commissural vein was formed by continuous differentiation of ground meristem cells into procambial progenitor cells, which started from a circular layer cell in a vascular bundle and progressed toward another vascular bundle in one direction (Fig. 6b). However, in rare cases, we observed a developing commissural vein that lacked periclinal cell division in its center. (Fig. 6c). This suggests that two commissural veins might have emerged from two neighboring longitudinal veins and been connected at the center.

In maize, although it was difficult to follow the entire process of commissural vein development, the initiation of the commissural vein occurred in one of the circular cell layer, just like in rice(Fig. 6d).

On the basis of these results, we propose a model of the differentiating process of the commissural vein in rice (Fig. 7). The initiation of the commissural vein occurred at a circular layer cell that was adjacent to both the metaxylem and phloem cells, and the initiation was observed in both the large vascular bundle (Fig. 6b) and the small vascular bundle (Fig. 6c). This initiation was associated with the development of the longitudinal vascular bundle, because it occurred only at stage V, in which metaxylem cells were formed. Therefore, developing metaxylem cells, and probably phloem tissue, might produce an intercellular signal(s) that leads the circular layer cell to differentiate into a procambial progenitor cell. Starting from the newly produced progenitor cell, a commissural vein is formed by continuous differentiation of the middle layer of the ground meristem cells into progenitor cells. This differentiation occurs mostly in one direction. In rare cases, however, two newly formed lines of procambial progenitor cells meet at the center. After carrying out studies with fluorescent markers, Scarpella and colleagues suggest that auxin transport restricted by AtPIN family proteins regulates procambium formation in Arabidopsis leaves (Scarpella et al. 2006; Sawchuk et al. 2007). One-directional and bi-directional procambium formation in commissural veins of rice leaves is similar to that in loop veins or higher order veins in Arabidopsis. It is, therefore, likely that directed auxin that is derived from developing metaxylem might be the signal for the differentiation of the procambium producing the commissural vein.

Fig. 7
figure 7

A model illustrating the formation of a commissural vein in rice. The differentiation into procambial progenitor cells forming the commissural vein commences at a circular layer cell (asterisk) that is adjacent to both a xylem vessel and a few phloem cells in a longitudinal vein. The differentiation signal appears to be transmitted to a vascular bundle sheath cell and then to a cell in the middle layer of the ground meristem. Differentiation of the procambial progenitor cell progresses in the middle layer of the ground meristem in one direction, toward the next longitudinal vein. After the differentiation of the procambium progenitor cell has been completed, periclinal cell division (arrows) commences from the procambial cell derived from the circular layer cell to form four vascular cells, and extends along the procambial progenitor cells. Finally, a commissural vein composed of four vascular cells is completed. LV large vascular bundle, SV  small vascular bundle, GM ground meristem, MX metaxylem vessel, PX protoxylem vessel, Ph phloem tissue, CL circular layer, VBS vascular bundle sheath

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In conclusion, the results of our studies showed that we could divide the developmental process of the large vascular bundles into six stages in rice and five stages in maize. We also found that the initiation of the commissural vein in rice arose in a circular layer cell that was in contact with both a metaxylem vessel and one or a few phloem cells in stage V longitudinal vascular bundles, including the large and small vascular bundles. In addition, directional differentiation of ground meristem cells into procambial progenitor cells was induced from the circular layer cell. These results suggested that cell–cell interaction was involved in differentiation into procambial progenitor cells. We also compared these results with those from maize plants and found that these differentiation processes are common in both monocotyledonous plants.