The Root Apex of Arabidopsis thaliana Consists of Four Distinct Zones of Growth Activities

Small size and simple anatomy is useful.

Arabidopsis root apices were analyzed in vivo and in vitro against a background of the above the maize information. As guidance for the information to follow, a representative root apex of a five-day-old Col-0 Arabidopsis seedling is shown in Figure 1, in which an epidermal trichoblast cell file is highlighted. This confocal micrograph of a live root stained with propidium iodide clearly shows the cell walls. In plant tissues, there is no movement of cells relative to each other. Therefore, cell growth measured for one cell layer is by definition also indicative for the other neighboring cell layers of the organ, unless cell division occurs which is not the case here. In earlier work,⁴³ we have introduced the stage of root hair bulging in trichoblasts as a new useful marker for time-related cellular development. Cells passing this critical developmental stage are extremely sensitive to both auxin⁴⁴ and ethylene.^43,45

During steady-state growth of Arabidopsis roots used in our experiments, a new cell reaches this stage of root hair bulging in each thrichoblast cell row every 30 minutes (see the video sequence at: http://webhost.ua.ac.be/fymo/Root.avi). Knowing the regular timing of development in the trichoblast cell file, it is easy to measure and calculate the course of cells throughout root apices. Obviously, the small size and simple anatomy of Arabidopsis roots makes this model object extremely useful for the detailed in vivo characterization of individual root zones of cellular activities as well as for their easy localization in root apices (Table 1).

Cell division is limited to the meristematic zone.

There are almost no reports that describe the basal (proximal) limit of the apical meristem in root apices of Arabidopsis. Classical cytological studies using pulse-labeled (usually with tritiated thymidine) samples did not include Arabidopsis as object of interest.⁴⁶ This species only acquired the status of model plant more recently.^1,13,14,47 Fortunately, current molecular biology techniques allow a direct approach to answer the question of meristem length. One can easily visualize the expression of proteins which are exclusively confined to dividing cells, such as cyclin B1 using (CYCB1;1) promoter-GUS lines.^48–50 Hauser and Bauer³⁰ used this rather elegant technique and reported that the proximal (basal) limit of the meristematic zone is situated at about 160 µm from the RCJ, and that the zone contained an average of about eight mitotic cells per cell file. There are several other reports, all of which did not specifically focus on the size of the meristem, but that did make use of the same CYCB1;1 transgenic line. All these data reveal invariably that the most proximal cell divisions are localized somewhere between 100 and 200 µm from the RCJ, depending on growth conditions and seedling age.^29,51–55 During our studies of the cytoskeleton in Arabidopsis roots,⁴⁵ we have performed a detailed analysis of the occurrence of mitotic and cytokinetic figures along the apex (Le J, unpublished data). Our observations are in accordance with the data published with the CYCB1;1 line, as we never detected preprophase bands, mitotic spindles or phragmoplasts farther than 200 µm from the RCJ.

Our values observed in vivo are in contrast to those indirectly computed from kinematic analysis of Arabidopsis root growth, localizing the proximal limit of the meristem between 488 and 713 µm from the RCJ, encompassing some 43 to 70 meristematic cells for one cortical cell file.^24,25,56,57 On the contrary, calculations performed on longitudinal root sections correspond well with data from work with the cyclin reporter gene constructs. Using the longitudinal section through the Arabidopsis root apex (Fig. 2) published by Ishikawa and Evans,³⁵ one can approximately calculate the number of cells in a cell file. In the left epidermal file, which is intact throughout this section, there are about 22 cells in the meristem (the first 200 µm from the RCJ) and about 20 cells in the transition zone (between 200–400 µm from the RCJ; for the determination of the basal limit of the transition zone see below). Kidner et al¹⁵ calculated the average number of dividing cells from longitudinal sections of 3 days old Arabidopsis root apices. These authors counted the number of cells within particular cell files from the initial cells towards the nondividing cells. They reported that the number of cells forming the apical root meristem varies between 18 (stele, atrichoblast) up to 35 (trichoblast) cells. From these data, it is clear that the indirect kinematic technique^2,24,25,57 shifts the basal border of the meristem into the transition zone or even beyond (see later). The problems arising with such kind of indirect analysis might be the consequence of noise-rich smoothening and curve-fitting procedures.

Thus, root cells leave the meristem somewhere between 150–200 µm from the RCJ (Fig. 1) in roots of Arabidopsis thaliana ecotype Col-0 seedlings grown in vitro at 23°C, 16 h/8 h light/dark cycle, on agarose for about five days. However, this value is not absolute for all Arabidopsis roots grown under other conditions, as the size of the meristem varies according to factors regulating plant development. During plant growth, the increase in the rate of root growth is accompanied by an enlargement of the apical meristem.²⁴ Also, it is known that apical root cells are monitoring signals arriving from older more proximally located cells.⁵⁸ Among these signals, sucrose and auxin are the most likely candidates. For instance, addition of 4.5% sucrose to the medium of growing Arabidopsis roots increased the number of dividing cells and enlarged the size of the apical meristem, shifting its proximal limit from about 162 to about 300 µm from the RCJ.³⁰

Similar to sucrose, auxin increased the size of root meristems, while addition of cytokinin resulted in a decrease in the size of apical meristems of Arabidopsis roots.²⁵ In accordance with this effect of cytokinin, genetically engineered tobacco plants with a reduced cytokinin content due to expression of cytokinin oxidase genes from Arabidopsis, showed substantially enlarged root meristems.^59,60 A more recent report confirmed that cytokinins control the exit of cells from the root meristem in Arabidopsis.⁶¹ Besides these signal-mediated changes in the size of the apical meristem, ectopically expressed mitotic cyclins are known to increase the size of the root meristem⁴⁸ while dominant-negative forms of Cdc2a kinase lower the population of dividing cells in Arabidopsis root apices.⁶² Therefore, these core cell cycle molecules appear to act as apparent targets for signal-transduction cascades regulating the cell cycle during growth and development of plant roots. In fact, tight correlations were reported between cell production rates and activities of cyclin-dependent kinases between roots of several Arabidopsis ecotypes.²¹

In spite of the wide availability and usage if the CYCB1;1 line, it is rather surprising that the basal border of the Arabidopsis root meristem is still an controversial issue in the current literature. Many authors simply ignore the transition (or the DEZ) zone and consider the onset of rapid cell elongation as the basal limit of the root meristem.^63–65 On the basis of the CYCB1;1 line, as well as of the careful analysis of the MAP4-GFP line (Le J, unpublished data), the proximal (basal) limit of the meristem is at about 200 µm from the RCJ. The subsequent growth zone, about 320 µm long, corresponds to the transition zone.

In the transition zone, the growth rate is very low and transformation from early post-mitotic to preelongation cells takes many hours.

At about 520 µm from the root cap junction, cell length increases noticeably (Fig. 1). This zone of rapid cell elongation will be discussed later. The cells distal to the zone of fast cell elongation (approximately between 200 to 520 µm from the RCJ) belong to the transition zone.

At 200 µm from the RCJ, cells enter the transition zone with an average cell length of 8 µm. During the subsequent 10 hours, their length increase is negligable reaching only 9 µm at 280 µm from the RCJ, although cell width increases from 14 to 16 µm. This section of the trichoblast cell file is not within the focus plane in the micrograph (Fig. 1). However, in the same cell file, a row of 17 cells covering the distance from 280 to 520 µm from the RCJ (distal to the arrowhead mark) can clearly be discerned: in that row, the elongation from 9 µm to 30 µm takes 8.5 hours. Furthermore, elongation is not homogeneously spread. In the the youngest six cells (three hours of development), no increase in cell length can be measured, while in the oldest six cells the growth in length is much more substantial. It is precisely in the latter part of the transition zone that trichoblast cells develop a central vacuole (upper asterisk in Fig. 3), while this feature is completely absent in the more distal part (younger cells, lower asterisk in Fig. 3) of the transition zone. In Figure 3, the arrowhead marks the end of the transition zone and the arrow the end of the elongation zone. This allows direct comparison with the (Fig. 1).

Cells in the proximal part of the transition zone are competent for the onset of fast cell elongation while cells in the distal part are competent to return back to the cell cycle activity. This finding coincides with the expression of cdc2 also in cells of the apical part of the transition zone (see Fig. 2F in ref. 66), suggesting that cells of the distal part of the transition zone maintain competence for cell division. All this renders the transition zone a kind of dynamic reservoir of developmentally plastic cells allowing rapid adjustment of both growth speed as well as direction of root growth according to demands of the actual environmental challenges.

As mentioned earlier, the development of trichoblast cell files mirrors somehow the development occurring in other cell types. Atrichoblasts are longer than trichoblasts (in general about 15%). This feature is seen throughout the transition zone. Vacuolization in this cell-type, as well as in the cortex cells, starts closer to the RCJ than in trichoblast cells, as illustrated in Figure 2. Cortex cells, on the other hand, are good markers for following increases in cell widths that occur in the transition zone. The final width of the cortex cells is only reached at the proximal end of the transition zone at about 520 µm from the RCJ. This is fully in accordance with the situation in maize root apices⁷ and can serve as another indication of the limit of the transition zone.

Based on kinematographic analysis of the root surface extension, several authors have defined the border between slow cytoplasmic growth and fast cell elongation arbitrarily as the point where the relative elemental growth rate reached 30% of its maximum value.^18,35,37,38 Incidentally, this indirect approach fits well with results of our in vivo cytological analysis reported here.

As a matter of fact, the concept of slow cell growth preceding the abrupt acceleration driving the fast cell elongation was confirmed also for Arabidopsis hypocotyl cells.⁶⁷ Here, cells elongate synchronously during the first 48 hours at a low rate, before the first cells at the base of the hypocotyl start their fast elongation. During the first 48 hours, these slowly elongating cells can be compared with cells passing through the transition zone. Thus, in the root apex transition zone, cell expansion is slow and occurs not only in the longitudinal (axial), but also to a certain extent in the transverse direction. At the basal limit of the transition zone, cells abruptly enter the phase of fast cell elongation and stop their widening. This corresponds exactly to the situation in maize root apices.⁵ The onset of rapid cell elongation is accompanied by impressive changes in structure and function of vacuoles and cell walls.

Transition-zone cells are flooded with sucrose unloaded from mature protophloem elements and undergo striking cell wall alterations.

In the growing root apex, maturation of the protophloem elements occurs within the transition zone, approximately at 250 µm from the root cap junction,^68,69 allowing local unloading of phloem-transported sugars into this growth zone.^68,69 As illustrated before, the central vacuole of trichoblasts develops only in the proximal part of the transition zone. This morphological characteristic is corroborated by the finding that the expression of the tonoplast aquaporin γ-TIP starts abruptly at the proximal border of the transition zone.⁷⁰

Concomitant with these changes in the cytoplasm, the cell wall undergoes specific adaptations. In vivo localization of XET action highlighted its maximal values at the proximal end of the transition zone.⁷¹ Xyloglucan endotransglucosylase/hydrolase (XTH) is an enzyme that modifies the cellulose-xyloglucan network, the load-bearing structure in plant cell walls. XTHs can cleave xyloglucans and subsequently rejoin the newly formed ends to available xyloglucan chains or oligosaccharides.⁷² This activity creates the opportunity for a turgor-pressure driven increase of the distance between two adjacent cellulose microfibrils, leading to growth. Cells within the transition zone are characterized by a progressive increase of the XET activity in their cell walls.⁷¹ This XET activity reaches its maximum values at the end of cell growth in three dimensions, at the basal limit of the transition zone, and heralds the onset of strictly polarized rapid cell elongation (no further cell expansion in width).⁷¹ In fact, the XET activity can be taken as a physiological marker of the proximal part of the transition zone in root apices of Arabidopsis. McCartney et al.²¹ reported the occurrence of a cell wall pectic (1 → 4)-β-D-galactan in the transition zone of Arabidopsis roots. This points again to fundamental changes in cell wall properties occurring at the border between the transition zone and the zone of fast cell elongation. In order to loosen cell walls effectively, the cellulose and xyloglucan network is also affected by expansins which are expressed and localized to cells of the transition zone too.^73,74 The idea of structural changes occuring in the cell wall at this ‘no return’ developmental point is further stressed by the detection of an increase in cellulose, xyloglucan and methylesterified pectins at the onset of fast cell elongation using FT-IR (De Cnodder T et al., unpublished results).

In a recent study, we have shown that cortical microtubules in the epidermis of Arabidopsis roots have a strictly transverse orientation at the basal limit of the transition zone.⁴⁵ It was reported that the cessation of radial expansion in postmitotic root cells of Arabidopsis was closely associated with the strict alignment of cortical microtubules into transverse arrays.⁷⁵ When cortical microtubules do not accomplish this redistribution, postmitotic cells fail to restrict their radial expansion and increase in width even throughout the elongation region as was described in bot1 and fra2 mutants of Arabidopsis.^75,76 Similar links between cortical microtubules and cell polarity were indicated by several other mutants of Arabidopsis.^77–79 However, other mutants showed a reduced cell polarity, with thicker and shorter root cells, although their cortical microtubules were arranged in highly ordered transverse arrays. In this regard, we can mention CORE mutants,⁸⁰ the kor mutant⁷⁵ and rsw4/rsw7 mutants.⁸¹ Evidently, besides ordered cortical microtubules, also the correct in muro localization of wall metabolic events is critical. In this respect, the cell wall has a pivotal role.^{75,76,83–86}

At the plasma membrane-cell wall interface,⁸⁷ COBRA could potentially be involved in establishing polarity of cell growth of postmitotic root cells.⁸⁸ COBRA is a member of glycosylphosphatidylinositol (GPI) anchored proteins, which are anchored to the outer plasma membrane leaflet and with putative interactions with cell wall molecules. COBRA mRNA levels were shown to be dramatically upregulated in cells located around 200 µm (see Fig. 4D and E in ref. 88) from the RCJ interpreted by the authors as rapidly elongating cells. Both, from distances from the RCJ as well as from the shapes of these cells (see Fig. 4D and E in ref. 88) it is obvious that COBRA mRNAs and proteins are located abundantly within the transition zone. Root cells of the cobra mutant have a reduced content of cellulose and fail the polarization of cell expansion as they expand too much in width and do not achieve proper elongation.⁸⁸ Nevertheless, mutant root cells reach normal volumes. This phenotype suggests that COBRA could be important for the acquisition and maintenance of the nongrowing status of cross-walls which is the most critical event in the polarization of postmitotic root cells during their switch into the rapid cell elongation.⁴²

In the zone of fast cell elongation, cell length increases by 300% in less than three hours.

In the Arabidopsis root, fast cell elongation starts when cells leave the transition zone and slows down when trichoblasts initiate outgrowths of root hairs.⁴⁵ In the example shown in Figure 1, cells increase their length from about 35 µm (arrowhead) to 135 µm (arrow) in two hours. There is some variability in the elongation rate, but the whole process is always terminated in less than three hours as can be seen in other cell files (Fig. 1). Along the root axis, the end of the fast elongation is situated at about 900 µm from the RCJ. In normal growing conditions, additional increase in cell length occurs up to about 1500 µm from the RCJ. Further on, the relative elemental growth rate of the root is not statistically different from zero.⁴⁵

Fast cell elongation is based on, and accomplished by, processes clearly different from the slow cell growth in the transition zone. In situ analysis of cell wall composition by FT-IR spectroscopy reveals a unique character for this part of the root (De Cnodder T et al., unpublished results). The β-glucosyl Yariv reagent was found to inhibit specifically the fast cell elongation.⁸⁹ It affects fucosylated arabinogalactan-proteins which are required for full cell elongation in root apices as deduced from the mur1 phenotype of Arabidopsis.⁹⁰ In the epidermis wall, the cellulose fibrils are oriented parallel and strictly transverse to the root axis.⁹¹ The cortical microtubules mirror this organisation and are aligned strictly parallel and transverse to the root axis.⁴⁵ The rate of cell elongation is inversely related to the endogenous ethylene concentration.⁴³ Under saturating conditions, the fast cell elongation is completely abolished, as is the residual elongation in the growth-ceasing zone, and cells never elongate beyond the length reached at the basal limit of the transition zone. For trichoblasts this is 35 µm, a length also reported for dwarfed phenotypes like the ctr1-1 mutant that expresses a constitutive ethylene response. On the contrary, it was found that cells have the potential to elongate beyond their normal length as trichoblasts elongated up to 200 µm in the absence of ethylene, a cell size also found in ein2-1, an ethylene insensitive mutant.⁴³ Fast elongation is also very sensitive to other hormone signals. In the root apices of the stunted plant 1 mutant of Arabidopsis, rapidly elongating root cells were affected while slowly expanding more apical cells were unaffected.^25,56 In this particular mutant, cell elongation defects are mediated via cytokinin-based signaling as cytokinin-treated wild type seedlings truly phenocopied this mutant.²⁵ Moreover, auxin is also known to preferentially affect the fast cell elongation.^8,44,92

The basal limit of the fast cell elongation zone is marked by the disturbance of the transverse arrays of the cortical microtubules.⁴⁵ This feature is evident when the fast elongation is blocked by a high dose of ethylene (for maize roots see ref. 92). However, the microtubule reorientation is not causally linked to the observed stop in cell elongation. Primary are events in the cell wall, including a rise in apoplastic pH, callose deposition and cross-linking events steered by reactive oxygen species.⁹³ The transit from pure cell elongation to differentiation is highlighted by the expression of a fucose-containing epitope in the cell wall of epidermal cells.⁹⁰ Furthermore, epidermal cells become symplastically isolated when leaving the fast elongation zone.⁹⁴ Another marker for the end of rapid elongation is the accumulation of myosin VIII along the cross walls of the epidermal cells exactly where in the trichoblasts root hairs start to develop rapidly (Verbelen et al., unpublished results).