• The development of the vasculature in the inflorescence stem and pedicel of two common Arabidopsis thaliana ecotypes is reported, describing the tissues and events in primary and secondary growth of the stem vasculature.
• Light and transmission electron microscopy was used to investigate the vascular system during plant growth; cellulose, lignin and callose were detected by cytochemistry.
• The innermost cortical layer differentiated as a starch sheath during primary growth. The outermost parenchymatous layers of the interfascicular region lignified the walls at the onset of fascicular cambium activity. The secondary structure formed during silique production. In the internodes, the interfascicular cambium was mainly produced by starch sheath cells. In the interfascicular arcs of the internode, the secondary xylem mainly consists of xylem parenchyma whereas in the bundle region, it comprises vessels. Secondary phloem and xylary fibres were produced in limited quantities. Medullary sheath and phloem cap cells also became lignified.
• The results show that the secondary vasculature develops in the stem and pedicel, and that, simultaneously, some primary tissues lignify, enhancing stem rigidity. Secondary vasculature is compared with the vascular alterations reported for some mutants.
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In flowering plants, vascular patterning is thought to depend on the combined action of signals which orientate the differentiation of each cell within the vascular system; however, the molecular nature of these signals is still largely unknown. The spectrum of possible signals ranges from the well known phytohormones, such as auxin and cytokinin (Fukuda, 1997), to not yet well defined positional signals coming from already differentiated cells and able to determine the fate of undifferentiated adjacent cells (van den Berg et al., 1995). During normal ontogenesis, the vascular tissues always differentiate at predictable, species-specific positions, suggesting that there exists an interaction between signal molecules and the genetic control of vascular patterning. However, the responses to wounding and to abnormal growth conditions show that vascular patterning also exhibits a certain flexibility (Aloni, 1987).
During primary growth, the vascular system differentiates in a continuum from root to stem, despite the different arrangement and type (simple or collateral) of vascular bundles in these two organs (Esau, 1965). In dicotyledons, secondary vascular patterning in the stem is normally associated with secondary patterning in the root, ensuring the coordinated development and structural contiguity of the shoot-root axis. Nevertheless, the origin of the vascular cambia which generate secondary vasculature differs between the two organs, with that of the stem partially deriving from the vascular procambium (Esau, 1965).
To understand better the genetic control of vascular patterning and differentiation, the most promising strategy seems to be the analysis of mutants. A number of mutants showing defects in the vasculature have been recovered in Arabidopsis. To interpret these defects, detailed anatomical knowledge of the various organs is necessary. However, a complete anatomical description of the inflorescence stem is still lacking, and the studies conducted to date have produced conflicting results. For instance, Dharmawardhana et al. (1992) have reported that the Arabidopsis stem ‘does not normally undergo secondary growth’, although it is extensively lignified during bolting. This finding is in contrast to reports of secondary vascular growth in the root (Dolan et al., 1993; Dolan & Roberts, 1995) and in the hypocotyl (Busse & Evert, 1999). Furthermore, the repeated removal of developing inflorescences has been demonstrated to result in cambial activity with secondary xylem formation in both shoot and root (Lev-Yadun, 1994).
Although sclerenchymatous arcs between primary bundles have been widely observed in the inflorescence stem of Arabidopsis (Przemeck et al., 1996; Turner & Somerville, 1997; Zhong et al., 1997), their origin remains unknown. In other Brassicaceae species, Metcalfe & Chalk (1950) reported that these arcs were, depending on the species, either primary or secondary in nature. In Arabidopsis, since the primary cortex is separated from the stele by a layer that some authors have interpreted as an endodermis (Fukaki et al., 1998) and the sclerenchymatous arcs lie within this layer, these arcs may be considered as part of the vascular cylinder and their differentiation may be part of the vascular patterning. Furthermore, in the stem of ifl1 mutant, an abnormal radial and axial distribution of the interfascicular fibres in these arcs has been reported (Zhong et al., 1997).
The aim of the present study was to provide a complete description of the origin and development of the vascular system in the inflorescence stem of Arabidopsis, which is crucial to analysing mutants with defects in vascular patterning and in the differentiation of specific components of the vasculature. Since vascular development might differ between ecotypes, given the genetic variability of Arabidopsis and its phenotypic plasticity during the reproductive phase (Pigliucci, 1997), the study was conducted on two of the most common ecotypes, analysing the entire reproductive phase (i.e. from the onset of bolting to silique dehiscence).
Materials and Methods
Plant material and growth conditions
Seeds of the ecotypes Wassilewskija (Ws) and Columbia (Co-O) were vernalized for 4 d at 4°C in continuous darkness and then sown on commercial soil. Seed germination and plant growth were performed in growth chambers (Heraeus VOTSCH, Balingen-Frammern, Germany) at 21°C, 80% rh and 16 h light/day (14 LUMILUX White lamps, 200 µmol m−2 sec−1). Plant growth lasted 40 d.
The entire stem of 1-cm-tall plants was fixed overnight in 1% glutaraldehyde-4% formaldehyde in 50 mM sodium phosphate buffer (pH 7.2). After washing for 30 min in the same buffer, the samples were dehydrated through a graded ethanol series and embedded in Technovit 7100 (Kulzer, Hereaus), according to the manufacturer. Transverse sections (TS) and longitudinal sections (LS) (5 µm thick) were taken using a Microm HM 330 microtome (Microm, Walldorf, Germany) and stained with 0.1% toluidine blue.
Floral pedicels and inflorescence stems, collected at various times after bolting (4, 8, 10, 14, 17, 22, 25, 31, 38, and 40 days), were dehydrated through an ethanol series, embedded either in Technovit 7100 or paraffin (Histo-Line Lab-O-Wax, melting point 56–58°C), transversely sectioned at 10 µm with a Pabish Topsuper S-150 microtome (Pabish, Germany) and stained with 0.005% aniline blue in 0.07 M K2HPO4 for detecting β-1,3-glucans (callose) deposition in sieve plates under fluorescent light. The sections were observed with a Zeiss Axiolab epifluorescence microscope (Zeiss, Jena, Germany) using a beam splitter (cut-off 395 nm), an excitation filter (353–377 nm) and a barrier filter (397 nm). The xylem was recognizable by lignin autofluorescence and the phloem by callose epifluorescence. At the same sampling dates, hand-cut TS and LS sections were stained with carmine-iodine green mixture to detect cellulose and lignin in the cell walls (Ricci, 1973). The sections were taken from explants of the apical, middle, and basal regions of the stem (apical explants cut starting from the base of the main inflorescence and basal explants cut starting from the level of rosette leaves; each explant 1.5 cm in length at day 4 and 2 cm at the other days).
Micrographs of sections from 15 to 35 specimens for each ecotype and each stem region were acquired in digital form with a SonyDXC-101P camera applied to an Axiophot microscope (Zeiss, Jena, Germany) and analysed using Optilab/pro 2.6.1 software (Graftek, Miramande, France).
Statistical differences between means were calculated with Student’s t-test.
Transmission electron microscopy
The explants of the apical, middle, and basal regions of the inflorescence stem were fixed in 3% glutaraldehyde in 0.2 M sodium phosphate buffer (pH 7.4). The samples were postfixed in 1% osmium tetroxide for 2 h, dehydrated through an ethanol series, and embedded in Epon 812 resin (Agar Aids Stanstead, Essex, UK). Ultrathin sections were cut with a diamond knife, stained with 2% uranyl acetate and 1% lead citrate and observed using a Zeiss-CEM 902 transmission electron microscope (Zeiss, Oberkochen, Germany) at 80 kV.
In both the Ws and Co-O ecotypes, eustele differentiation was observed in the 1-cm tall stems and, at the stage of anthesis of the first flower, in the 5-cm tall stems and in the pedicel.
In the apical region of the 1-cm-tall stems of both ecotypes, 7–8 bundles, arranged in the eustelic structure, are already evident, and each bundle is capped by several, highly hypertrophic and thin-walled cells (Phc in Fig. 1a). These phloem-cap cells are located between the protophloem (Pph in Fig. 1a) and the innermost cortical layer, which has already assumed the aspect of a sheath surrounding the stele, since its cells are larger than those of the outermost cortical layers. In both ecotypes, the protophloem consists of very few sieve tubes that have already differentiated; the metaphloem is still mostly meristematic (Mph in Fig. 1a). The metaxylem consists of both expanded cells, still in the process of differentiating, and of smaller radially aligned meristematic cells (Mx in Fig. 1a). The protoxylem consists of a few vessels, with thick lignified walls (Px in Fig. 1a). The interfascicular regions, also known as ‘medullary primary ray regions’, consist of parenchymatous cells that are heterogeneous in shape, and, occasionally, also of meristematic cells.
In the basal region of the inflorescence stem the monolayered sheath surrounding the stele contains amyloplasts in its cells, thus assuming the characteristics of a starch sheath (Fig. 1b–c). In the interfascicular region, the cells are still parenchymatous; however, in the Ws ecotype, those cells near the bundles, and belonging to the outermost layers of the region, showed densely stained deposits at the corners (Fig. 1d). Electron microscopy revealed that these deposits mostly consisted of cellulose in the wall (Fig. 1e) and partly of cytoplasm and mitochondria near the plasmalemma. In both ecotypes, near the nodes, bundles consisting of 2–3 merging bundles were visible along the flanks of the gaps. These merging bundles were first observed just below the node. As a consequence of this merging process, the stele exhibits apparently fewer bundles. In the merged bundles, the protoxylem poles are always separated.
At the stage of anthesis of the first flower (4 d after bolting, 5-cm-tall stems), although the two ecotypes did not differ significantly in terms of the diameter of the apical region of the stem (620.3 ± 20.4 µm and 572.8 ± 18.8 µm for Ws and Co-O, respectively), the middle and basal regions were significantly thicker in the Ws plants (middle region: 724.5 ± 5.4 µm in Ws; 587.7 ± 17.5 µm in Co-O, P < 0.01; basal region: 721.8 ± 23.9 µm and 600.6 ± 18 µm, respectively, P < 0.05). In the apical region of the Ws stems, more phloem-cap cells were observed than in the 1-cm-tall stems; furthermore, several metaphloem cells had already differentiated and become radially aligned with the protophloem (Fig. 1f). In the metaxylem, lignified cells, expanded and thin-walled cells, and meristematic cells were all observed (Fig. 1f). In the 3–4 outermost cell layers of the interfascicular region (arc), the thickening at wall corners is more conspicuous than in the 1-cm-tall stems and was observed in cells along the entire interfascicular arc. In the basal region of the Ws stem, the metaphloem is almost totally differentiated, as indicated by the presence of callose plugs in the sieve tubes, whereas the metaxylem is still differentiating (Fig. 2a). In the Co-O stem, the cellulose deposition at wall corners in the interfascicular arcs and the differentiation of the primary xylem and phloem in the bundles is delayed in comparison with the Ws stem. In neither of the ecotypes do the cell walls of the interfascicular arcs show autoflorescence (Fig. 2b).
In the pedicel of both ecotypes, at the receptacle base (i.e. near the flower), the procambium appears as a ring in TS. The procambium has produced discrete vascular bundles separated by interfascicular ray parenchyma (Fig. 2c). The protoxylem differentiates closer to the receptacle base than does the ray parenchyma. The stele of the pedicel is nearly identical in structure to that of the apical stem of the 1-cm-tall plants, except for a higher number of meristematic cells in the interfascicular regions and a reduced size and quantity of bundles (up to 3–4) (data not shown).
Secondary vascular structure
For the histological analysis of the secondary vascular structure in the stem, three stages of reproductive growth after the anthesis of the first flower were defined: stage 1, the stage at which the first silique differentiated on the inflorescence; stage 2, the stage at which immature siliques appeared on the infructescence, yet apical floral buds were still present; and stage 3, the stage at which all siliques were dehiscent, with only very few apical buds still present.
In both ecotypes, stage 1 was reached 8 d after bolting. At 10 d, some green siliques were observed on the stem (onset of stage 2); at 22 and 25 d after bolting (Ws and Co-O, respectively), all the siliques were green (end of stage 2). Stage 3 was reached at 31 (Co-O) and at 38 (Ws) d after bolting. At this stage, the Ws plants were not significantly taller compared with stage 2, whereas the Co-O plants had increased significantly in height. In the Co-O plants, the diameter of the apical and middle regions was significantly lower compared with the Ws plants (P < 0.01).
At stage 1, in the middle and basal stem regions of both ecotypes, the cells of the 3–4 outermost layers of the interfascicular arcs (i.e. those cells which previously showed cellulose thickening) have a lignified wall (Fig. 2d), with a weak fluorescence signal (Fig. 2e, in comparison to Fig. 2b). As a consequence of lignin deposition, the stele, circled by the nonlignified starch sheath (Fig. 2d), shows bundles connected by lignified arcs (Fig. 2e). At this stage, in the Ws stems, the first vessels of the secondary xylem appear; these vessels, produced by the fascicular cambium in the bundles, are smaller than those of the metaxylem (Fig. 2f). In the Co-O plants, the first vessels of the secondary xylem often appear later (i.e. at the onset of stage 2).
At the onset of stage 2, in both ecotypes, the lignification of the interfascicular arcs becomes conspicuous along the entire stem (Fig. 2g), and the cells of the 3–4 outermost layers appear to have become extraxylary fibres, as shown by the reduced lumen, long spindle-like shape and simple pits in the wall. The radial extension (mean length × width in TS) of the xylem in the basal-stem bundles is 45.5(2.6) × 96.2(7.1) µm and 32.6 (1.4) × 81(4.5) µm for Ws and Co-O, respectively, whereas for the phloem it is 25.3(1) × 87.9(2.7) µm and 22.6(1.6) × 39.6(1.7) µm for Ws and Co-O, respectively.
At this time, the interfascicular cambium was observed at the nodal region of the basal stem, between merging bundles (Fig. 2h) (referred to as the ‘nodal if-cambium’). It is contiguous with the fascicular cambium of the bundles, which continues to produce secondary xylem. The nodal if-cambium was produced by periclinal and oblique divisions in nonlignified medullary ray and phloem parenchyma cells (Fig. 2h); in no case the starch sheath cells divide to contribute to the formation of the nodal if-cambium. Although the nodal if-cambium produces a limited number of secondary xylem and phloem elements (Fig. 2i), they are sufficient to close the gaps. After the nodal if-cambium has begun to produce these elements, another interfascicular cambium can be observed in the basal stem, specifically, in the wide interfascicular arcs of the internodal regions (referred to as ‘internodal if-cambium’). Near the bundle, it is produced by the same cell types as the nodal if-cambium and by starch sheath cells (Fig. 2j, inset), whereas far from the bundle, it is produced by starch sheath cells only (Fig. 2j). These sheath cells produce the internodal if-cambium by oblique or periclinal divisions (Fig. 2j). The division of the starch sheath cells extends from the bundles until it occupies the entire interfascicular arc (Fig. 2j). As a result of internodal if-cambium activity, small groups of radially aligned derivatives are produced externally to the extraxylary (primary) fibres of each arc. At the onset of stage 2, the longitudinal sections showed conspicuous differences between the products of the fascicular cambium and those of the internodal if-cambium. The vascular bundles retain the shape of the primary structure, but contain more xylem and phloem, due to fascicular cambium activity (Fig. 3a). In the interfascicular arc, external to the primary fibres, the internodal if-cambium produces cells that are short, very heterogenous in shape, and already partly lignified (Fig. 3b); these cells are interpreted to be secondary xylem parenchyma.
At the end of stage 2, in both ecotypes, the internodal if-cambium continues to form mainly secondary xylem parenchyma (Fig. 3c–e), whose cells are shorter than the vessel elements and have a thinner wall that is not always uniformly lignified. Although these cells vary in size and shape, most are larger than the vessels overall (Fig. 3e–f, in comparison). The internodal if-cambium also produces thin-walled cells which in turn differentiate into a few sieve elements (Fig. 3c), apparently following an endocytogenic growth pattern (Miller, 1980) (Fig. 3e). The fascicular cambium continues to produce secondary xylem mainly comprising vessels (Fig. 3d,f).
In all stem regions, some phloem-cap cells have lignified walls, a wide lumen in TS (Fig. 3g, arrow) and are very short in LS. Based on these characteristics, these cells (previously termed as phloem fibres by Zhong et al., 1997) are better described as fibre-sclereids of the primary phloem. At the same time, some of the cells produced by the internodal if-cambium near the bundles shows conspicuous lignification and, consequently, a very reduced lumen at maturity (Fig. 3g, circles). These cells are interpreted as xylary (secondary) fibres. In the interfascicular arc, some cells of the medullary sheath (i.e. the outermost layer of the central core of the pith) have a lignified wall yet remain wide (Fig. 3c, arrowhead) and short. Some medullary sheath cells surrounding the protoxylem pole in the bundle have a lignified wall, a reduced lumen, and a spindle-like shape.
At stage 3, in both ecotypes, the secondary vascular structure was observed along the entire stem. Although the protoxylem poles of the eustele bundles are still detectable, in the internodal regions, the secondary activity of the two cambia (i.e. fascicular and internodal if-cambia), which mainly produce xylem, greatly alters the general appearance of the vasculature. By this stage, the radial extension of the xylem in the bundles of the basal internode was significantly greater (P < 0.01 and P < 0.05 for Ws and Co-O, respectively) than that observed at the onset of stage 2 (93(4.2) × 142.4(5.5) µm and 52.4(2.8) × 94.9(6.2) µm for Ws and Co-O, respectively). By contrast, the radial extension of the phloem does not significantly change (24.2(0.9) × 96.3(3.2) µm and 24.9(1) × 68.4(4) µm for Ws and Co-O, respectively). Furthermore, in the Ws ecotype, the radial extension of the interfascicular arc increases from 40.9(3.1) µm at the onset of stage 2 to 71.5(6.2) µm at stage 3; in the Co-O ecotype, it increases from 35.4(1.9) µm at the onset of stage 2 to 56.5(3.1) µm at stage 3. Since there is also a consistently small amount of secondary phloem in the interfascicular regions, the highly significant increase (P < 0.01 for both ecotypes) in the radial extension of the lignified arc can be mainly attributed to the production of secondary xylem parenchyma.
At stage 3, the pedicel shows the secondary vascular structure (Fig. 3h) whilst suber and lenticel differentiation (Fig. 3i) have occasionally occurred at the stem surface.
Brassicaceae are characterized by lignified interfascicular regions (Metcalfe & Chalk, 1950). In some genera, this sclerenchymatous tissue does not contain vessels, though it is produced by the cambium. Therefore, it can be interpreted either as a secondary xylem devoid of vessels or as a lignified medullary ray tissue produced by the cambium. According to Zhong et al. (1997), the lignified arcs joining the bundles in Arabidopsis are interfascicular fibres that are extraxylary (primary) in nature. According to Lev-Yadun (1997), they are fibres of the secondary xylem (i.e. xylary fibres). Finally, according to Turner & Somerville (1997), they constitute a sclerified parenchyma separating the primary bundles. Our analysis shows that, when the secondary vascular structure is fully developed in stem and pedicel, the interfascicular arc consists of a mixed population of cells, only in part of cambial origin, but all, except the secondary phloem, characterized by strong lignification. Thus, in the mature plant, the lignified portion of the interfascicular arc is in part primary and in part secondary in nature, and both components contribute to stem stiffness.
The cellulose deposition at the wall corners of the cells of the outermost layers of the interfascicular region preceded the formation of the first elements of the secondary xylem produced by the fascicular cambium. This latter event occurred simultaneously with the lignification in the cells with cellulose deposits, which were then transformed into extraxylary fibres. The secondary modification of these primary cells thus seems to be coordinated with the differentiation of the secondary structure. Turner & Somerville (1997) observed a uniform wall thickness in the modified cells of the outermost layers of the interfascicular region. They also showed a preferential deposition of wall material in the cell corners in irx2, a mutant deficient in cellulose deposition in the secondary cell wall (Turner & Somerville, 1997). From our analysis, it appears that the formation of the cellulose wall thickening starts near the bundles, when the bundles are still in a primary state, and extends along the entire interfascicular arc. It is thus possible that the cellulose deposition in the irx2 mutant is slower or delayed with respect to that in the wild type and that the modality of deposition is not affected. It has been reported that interfascicular parenchyma shares common differentiation events with vascular cells of procambial and cambial origin (Sachs, 1981). Since cellulose deposition begins near the bundles, it might be triggered by auxin and/or other diffusible signals originating from the eustelic bundles. Alternatively, it is possible that a positional signal from the bundles induces the nearby cells to differentiate into a tissue with cellulose deposits in the wall corners and that this process is repeated until it embraces the entire interfascicular region.
This study revealed the activity of a continuous cambial ring in the stem of Arabidopsis. Fascicular and interfascicular cambia, and their derivatives, have been previously observed in Arabidopsis mutants, such as ifl1/rev-1 (Talbert et al., 1995; Zhong & Ye, 1999) and Atpin1 (Galweiler et al., 1998). Cambial activity resulting in the formation of considerable amounts of secondary xylem has also been obtained using the procedure of repeated cuttings of Arabidopsis stem (Lev-Yadun, 1994). We found that an if-cambium first appears between merging bundles at the nodal region and later in the wide interfascicular arcs of the internodal region. In plants undergoing secondary vascular growth, the if-cambium is involved in closing the gaps, which is associated with the early breaking of the leaf trace (Esau, 1965). The nodal if-cambium is produced by parenchyma cells near the bundles, and its activity leads to the production of very limited amounts of secondary xylem and phloem. The internodal if-cambium is more productive than the nodal if-cambium and is mainly active in producing secondary xylem parenchyma. Furthermore, it has a more complex origin. Specifically, it is produced by the division of the same types of cells as the nodal if-cambium, but situated in the sheath cells surrounding the primary vasculature. Although Fukaki et al. (1998) referred to this sheath as an endodermis, electron microscope analysis shows that it is undoubtedly a starch sheath. Starch sheath cells have also shown to undergo division in other developmental processes, such as adventitious organogenesis in cultured tomato leaves (Coleman & Greyson, 1977). Furthermore, the presence of the starch sheath has been recently reported in Arabidopsis; here a mutation of the SCARECROW gene results in the loss of the normal starch sheath and an inability of the shoot to sense gravity (Wysocka-Diller et al., 2000). Thus the starch sheath, at least in Arabidopsis, may be essential for both shoot gravitropism and internodal if-cambium formation.
The secondary xylem parenchyma produced by the internodal if-cambium, together with the extraxylary fibres, become the main components in the lignified arc at maturity. However, a few xylary fibres, produced by the internodal if-cambium, are also present. In the stem of the ifl1 mutant, an abnormal distribution of the interfascicular fibres has been reported (Zhong et al., 1997). In the stem of the rev-1 mutant, the interfascicular fibres do not appear as highly lignified as in the wild type (Talbert et al., 1995). It is thus possible that the IFL1/REV gene (Zhong & Ye, 1999; Ratcliffe et al., 2000) controls the activity of at least the internodal if-cambium.
Finally, the presence of suber revealed the activity of a phellogen (i.e. cork cambium), the other lateral meristem contributing to plant secondary growth (Esau, 1965). Phellogen has been previously observed in the root and hypocotyl of Arabidopsis (Dolan et al., 1993; Busse & Evert, 1999). This observation shows that the Arabidopsis inflorescence stem has full secondary growth (i.e. not only secondary vascular growth but also periderm formation) and that this event is not a peculiarity of a specific ecotype.
The authors wish to thank Professor Maria Grilli and Dr Antonella Canini of the University of ‘Tor Vergata’ (Italy) for their help in the electron microscopy. The research was supported by funds from Ministero dell’Università e della Ricerca Scientifica e tecnologica (Cofin ’98), provided to MMA, and from Ministero delle Politiche Agricole e Forestali, Piano Nazionale ‘Biotecnologie Vegetali’, Progetto n.352, Area 8, provided to MMA.