**3. Vascular tissue development and regeneration in mechanically stimulated inflorescence stems of** *Arabidopsis*

In this paragraph, we will describe in detail the transition from primary to secondary tissue architecture in inflorescence stems of *Arabidopsis* as the important step in obtaining a suitable model for secondary vascular tissue analysis, following the temporal and spatial changes during vascular cambium ontogenesis, xylem formation, and vascular tissue regeneration in weight-induced *Arabidopsis*.

#### **3.1. Ontogenesis of vascular cambium**

Ontogenesis of vascular cambium is correlated with temporal and spatial changes on the stem circumference. Usually, formation of a closed ring of cambium is preceded by dedifferentiation of parenchyma cells into cambial cells and the so-called interfascicular cambium development. This process is commonly observed in young woody plants during their secondary growth [12]. It has been confirmed by histological analyses that the first dedifferentiated parenchyma cells are localized next to the vascular bundles in the early stages of the interfascicular cambium development [12]. With the time, the regions of dedifferentiating parenchyma cells are extended and finally enclosed as continuous ring on the stem circumference. The mechanism of these changes is still not clarified. The basic question is which of the cellular events trigger the parenchyma cell dedifferentiation?

In mechanically stimulated *Arabidopsis* stems, vascular cambium develops from fascicular cambium and interfascicular cambium bands (**Figure 2**). Fascicular cambium develops as a primary meristematic tissue in vascular bundles, localized in the inner parts of immature stems characterized by primary tissue architecture. The vascular bundles are separated by interfascicular parenchyma bands with nonpericlinally dividing parenchyma cells (**Figure 2A** and **B**). Outside these regions, few layers of cortex and single layer of the epidermis are situated. Middle parts of the stems consisted of the enlarged, thin-cell wall pith parenchyma cells. One- or few-layer supporting tissue with characteristic thick-cell wall interfascicular fibers plays mechanical function in immature stems (**Figure 2B**). In immature stems of *Arabidopsis*, 6 days after weight application, the architecture of the basal parts of such stems diametrically changes. At the beginning of the secondary growth, interfascicular cambium develops in the interfascicular regions of stems, as a consequence of parenchyma cell dedifferentiation (**Figure 2C** and **D**). Interestingly, the most inner layer of interfascicular parenchyma cells dedifferentiates into the interfascicular cambium. Typically, it is a single layer of parenchyma

**3. Vascular tissue development and regeneration in mechanically** 

In this paragraph, we will describe in detail the transition from primary to secondary tissue architecture in inflorescence stems of *Arabidopsis* as the important step in obtaining a suitable model for secondary vascular tissue analysis, following the temporal and spatial changes during vascular cambium ontogenesis, xylem formation, and vascular tissue regeneration in

**Figure 1.** Intrusive growth of the fusiform cambial cells in mechanically stimulated *Arabidopsis* stems. (A) Schematic visualization of two intrusively growing cells ((1) two neighboring cells with still non-intrusively growing ends, (2) beginning of the intrusive growth of the opposite ends, (3) advanced intrusive growth along the radial cell walls of the cells). Asterisks indicate neighbor cells and their intrusively growing ends. (B–D) Intrusively growing fusiform cambial cells in temporal steps corresponding to situation visualized in (A). (B–D = longitudinal tangential sections through *Arabidopsis* stems; Poly/Bed 812 resin sections stained with the Periodic Acid—Schiff's (PAS reaction); bar, 10 μm).

Ontogenesis of vascular cambium is correlated with temporal and spatial changes on the stem circumference. Usually, formation of a closed ring of cambium is preceded by dedifferentiation of parenchyma cells into cambial cells and the so-called interfascicular cambium development. This process is commonly observed in young woody plants during their secondary growth [12]. It has been confirmed by histological analyses that the first dedifferentiated parenchyma cells are localized next to the vascular bundles in the early stages of the interfascicular cambium development [12]. With the time, the regions of dedifferentiating parenchyma cells are extended and finally enclosed as continuous ring on the stem circumference. The mechanism of these changes is still not clarified. The basic question is which of the cellular events trigger the parenchyma cell

**stimulated inflorescence stems of** *Arabidopsis*

weight-induced *Arabidopsis*.

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dedifferentiation?

**3.1. Ontogenesis of vascular cambium**

**Figure 2.** Transition from primary to secondary tissue architecture in weight applied inflorescence stems of *Arabidopsis*. (A) Schematic visualization of the tissue arrangement in immature inflorescence stems of *Arabidopsis*. (B) Cross section through the basal parts of stem with the primary tissues—vascular bundles with fascicular cambium are separated by the interfascicular parenchyma. The most inner layer of interfascicular parenchyma cells will dedifferentiate into cambial cells (asterisks). (C) Schematic visualization of the secondary vascular tissues with closed ring of vascular cambium on stems circumference. (D) Layer of periclinally dividing interfascicular cambial cells as a part of the ring of vascular cambium. The first developed vessels are indicated by asterisks (lignin in the secondary cell walls stained with 0.05% Toluidine Blue O aquatic solution). B and D, handily made cross sections through basal part of stems (VB, vascular bundle; IFP, interfascicular parenchyma; *iff*, interfascicular fibers; *co*, cortex; *pp*, pith parenchyma; *fc*, fascicular cambium; *ifc*, interfascicular cambium); bars, 20 μm.

cells localized between vascular bundles. Finally, during the transition from the primary to the secondary tissue architecture of *Arabidopsis* stems, fascicular and interfascicular cambium forms fully enclosed ring of vascular cambium on stem circumference (**Figure 2D**).

The whole process of cambium ontogenesis is strictly correlated with such cellular events as elevated auxin response in interfascicular parenchyma, polarity of parenchyma cells dedifferentiating into the cambium, their periclinal divisions, and changes of their cell wall components [7]. The most spectacular seems to be correlations between auxin response and tissue polarity during cambium ontogenesis in analyzed *Arabidopsis* stems. Already in the first few days, auxin concentration distinctly arises in the dedifferentiating parenchyma cells. At the early stages of the interfascicular cambium development, maximum auxin concentration is detected in parenchyma cells localized in the nearest neighborhood of vascular bundles, whereas in the later stages of this process, the zone of the cells with elevated auxin response is gradually extended toward the middle parts of the interfascicular regions, in the next few days after weight application [7]. Polarity of the interfascicular parenchyma was monitored by the PIN-FORMED1 (PIN1) protein localization in differentiating cells. The PINs are well-known auxin transport proteins involved in the cellular efflux of auxin and polar auxin transport in plant tissues [53]. In many developmental processes, the establishment of local PIN-dependent auxin gradient in cells is strictly correlated with cellular divisions and developmental reprogramming [54, 55].

During analyzed process of cambium ontogenesis, tissue polarity is rapidly established in *Arabidopsis* stems. Amazingly, polarity of interfascicular parenchyma is indicated by polar localization of PIN1 auxin transport protein, which localizes at the basal plasma membranes of differentiating cells [56]. It has been documented that the protein appears in the basal plasma membranes of dedifferentiating parenchyma cells, not previously found in parenchymatic cells of immature mechanically noninduced *Arabidopsis* stems [7]. Moreover, both of the events—elevated auxin response and tissue polarization—are accompanied by periclinal divisions of the parenchyma

**Figure 3.** Periclinal divisions of interfascicular parenchyma cells and interfascicular cambium development in weightinduced *Arabidopsis* stems. (A) Schematic visualization of the temporal changes in interfascicular parenchyma regions with gradually extended zone of periclinally dividing cells (1–4 = four steps of the changes from vascular bundles, dark grey; zone of dividing parenchyma cells, grey; nondivided parenchyma cells, white; arrows indicate the direction of changes). (B) Periclinal divisions of the interfascicular parenchyma cells in the neighborhood of the vascular bundle (arrows); Poly/Bed 812 resin section stained with the periodic acid-Schiff's (PAS reaction); *vb*, vascular bundle; *ifr*, interfascicular region; bar, 20 μm.

cells (**Figure 3**). Divisions are temporarily correlated with the cellular events mentioned above and maintained in space. Namely, the first periclinally divided cells appear in the neighborhood of vascular bundles (**Figure 3A** and **B**), but later the zone of dividing cells slowly extends toward the middle part of interfascicular regions. In consequence, parenchyma cells dedifferentiate into cambial cells, which definitely changes architecture of the interfascicular regions and decides about development of the interfascicular cambium (**Figure 3A**). According to the obtained results, it is tempting to conclude that auxin plays the most important role during cambium ontogenesis in *Arabidopsis* stems. Auxin seems to be a primary signal for cellular fate reprogramming and a crucial clue for stimulation of the dedifferentiational process in the interfascicular parenchyma zones.

In the described model, vascular cambium could be classified as "functioning" meristematic tissue, which actively produces cambial derivatives. Differentiation of cambial derivatives is a consequence of numerous periclinal divisions of fusiform cambial cells. Finally, the maturation of the cambial derivatives into secondary vascular tissue elements supported functionality of this meristematic tissue in the present model. The sequence of the changes could be useful for all comparative analysis of the cambium ontogenesis and xylogenesis both in *Arabidopsis* model system and the analogical mechanisms studied in woody plants. Thus, the mechanically stimulated *Arabidopsis* model with fully functional cambial meristem could help us in addressing the elusive vascularization mechanisms observed in the woody plants.

### **3.2. Secondary xylem formation in** *Arabidopsis* **stems**

cells localized between vascular bundles. Finally, during the transition from the primary to the secondary tissue architecture of *Arabidopsis* stems, fascicular and interfascicular cambium

The whole process of cambium ontogenesis is strictly correlated with such cellular events as elevated auxin response in interfascicular parenchyma, polarity of parenchyma cells dedifferentiating into the cambium, their periclinal divisions, and changes of their cell wall components [7]. The most spectacular seems to be correlations between auxin response and tissue polarity during cambium ontogenesis in analyzed *Arabidopsis* stems. Already in the first few days, auxin concentration distinctly arises in the dedifferentiating parenchyma cells. At the early stages of the interfascicular cambium development, maximum auxin concentration is detected in parenchyma cells localized in the nearest neighborhood of vascular bundles, whereas in the later stages of this process, the zone of the cells with elevated auxin response is gradually extended toward the middle parts of the interfascicular regions, in the next few days after weight application [7]. Polarity of the interfascicular parenchyma was monitored by the PIN-FORMED1 (PIN1) protein localization in differentiating cells. The PINs are well-known auxin transport proteins involved in the cellular efflux of auxin and polar auxin transport in plant tissues [53]. In many developmental processes, the establishment of local PIN-dependent auxin gradient in cells is strictly correlated with cellular divisions and developmental reprogramming [54, 55].

During analyzed process of cambium ontogenesis, tissue polarity is rapidly established in *Arabidopsis* stems. Amazingly, polarity of interfascicular parenchyma is indicated by polar localization of PIN1 auxin transport protein, which localizes at the basal plasma membranes of differentiating cells [56]. It has been documented that the protein appears in the basal plasma membranes of dedifferentiating parenchyma cells, not previously found in parenchymatic cells of immature mechanically noninduced *Arabidopsis* stems [7]. Moreover, both of the events—elevated auxin response and tissue polarization—are accompanied by periclinal divisions of the parenchyma

**Figure 3.** Periclinal divisions of interfascicular parenchyma cells and interfascicular cambium development in weightinduced *Arabidopsis* stems. (A) Schematic visualization of the temporal changes in interfascicular parenchyma regions with gradually extended zone of periclinally dividing cells (1–4 = four steps of the changes from vascular bundles, dark grey; zone of dividing parenchyma cells, grey; nondivided parenchyma cells, white; arrows indicate the direction of changes). (B) Periclinal divisions of the interfascicular parenchyma cells in the neighborhood of the vascular bundle (arrows); Poly/Bed 812 resin section stained with the periodic acid-Schiff's (PAS reaction); *vb*, vascular bundle; *ifr*,

interfascicular region; bar, 20 μm.

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forms fully enclosed ring of vascular cambium on stem circumference (**Figure 2D**).

Reprogramming of the gene expression that accompanies xylogenesis and transdifferentiation of mesophyll cells into tracheary elements was extensively studied in *in vitro* cultures of zinnia (*Zinnia elegans*) [57, 58]. However, the lack of the cambium stage in this experimental system prevents us from deciphering the role of cambium in wood formation. Temporal gene expression pattern accompanies dedifferentiation of cambial cells into cambial derivatives, but their maturation into different types of tracheary elements is poorly characterized. Thus, numerous efforts have been focused on the identification of master regulatory genes required for this transition and revealing the key components of the vascular-differentiation-involved genetic network [48, 59].

In *Arabidopsis* "cambial" model, vascular cambium reveals basic features of functioning cambium important in the following stages of xylogenesis. Periclinal divisions of the fusiform cambial cells lead to the development of secondary xylem derivatives in the early stage of xylogenesis. Changes in later stages of xylogenesis are correlated with maturation of cambial derivatives into tracheary elements and secondary vascular xylem development (**Figure 4**). During this process, such recognizable tracheary elements as vessels, fibers, or tracheids develop and create the layer of secondary xylem. Vessels are easily recognized, because of some diagnostic features such as secondary cell wall and open perforation plates on the opposite ends of the vessel members (**Figure 4C**). Vessels are arranged in threads of longitudinal strands in the vascular tissue. Amazingly, in the present *Arabidopsis* model, impressive variety of tracheary elements is detected, not previously documented in analyzed hypocotyls [37, 38] or adult stems of *Arabidopsis* [39–41].

**Figure 4.** Secondary xylem in the weight stimulated stems of *Arabidopsis*. (A) Secondary xylem elements, like vessels and fibers, are produced from cambial derivatives after numerous periclinal divisions of fusiform cambial cells. Cortex parenchyma is visible outside the secondary vascular tissues. (B) Schematic visualization of the tissue arrangement in stem and localization of the tissues showed in (A) is indicated by the square. (C) Vessel strand developed parallel to longitudinal axis of stem. Characteristic patterning of the secondary cell wall (arrow) and perforation plates developed on the opposite apical-basal ends of neighboring vessel members (circle) determines the most diagnostic features for this type of tracheary elements (A and C, bright-field images in a confocal laser-scanning microscope; *fb*, fibers; *sxv*, secondary xylem vessels; *vc*, vascular cambium; *co*, cortex); bars, 50 μm (A); 20 μm (C).

Patterning of vascular tissue and variety of tracheary elements developed as a dynamically operating water-conducting system and was extensively studied in the woody plants [13, 14]. However, mechanism regulating xylogenesis at cellular and molecular levels remains unclear, and many questions are unanswered. For example, differentiation of tracheids as a type of tracheary elements commonly found in trees, but for the first time detected in mechanically stimulated *Arabidopsis*, led to important conclusions about the involvement of the artificial weight in wood formation. Following stages of xylogenesis involving formation of the variety of tracheary elements, such as recognized tracheids, will be helpful in future analysis.

#### **3.3. Regeneration of vascular tissue in wounded** *Arabidopsis* **stems**

In 1981, Sachs postulated canalization hypothesis according to which vasculature patterning is based on the positive feedback loop between auxin flow and cellular polarity. Consequently, in the primary uniform tissue, cellular auxin transporters emerge as the so-called auxin channels that transport the hormone through the tissue in the polar direction. Emergence of auxin channels is correlated with establishment of cellular polarity inside these specific auxin transport routs. Finally, new vessels develop directly along the auxin channels. Canalization hypothesis is strongly supported by many classical experiments with the incised plants, i.e., by wounding or grafting, which shows that emergence of auxin channels is correlated with increased auxin response and tissue repolarization [1, 2, 4, 5]. It is well documented that initially broadly elevated auxin response in wounded tissues is gradually restricted to narrow auxin channels, in which auxin level is still very high [4]. The obtained results showed that patterning of vascular tissue, explicitly visible during regeneration and new vasculature development, is dependent on new ways of canalized auxin flow.

Well-functioning vascular cambium plays the most important role for the secondary growth in the woody plants, both secondary xylem formation and stem thickness [14, 21, 22, 60]. Many results revealed an important role for this meristematic tissue during vasculature regeneration process. For decades analysis of vascular patterning and incised vascular cambium regeneration was restricted mainly to trees [61–63] because these woody plants undergo secondary growth with enlarged amount of secondary xylem (wood) and active cylinder of vascular cambium [64]. Studies were based mainly on the histological analysis, thus limited only to the final effects of regeneration. Thus, it was impossible to analyze vasculature regeneration, including vascular cambium, on the cellular and molecular levels. Some experimental studies on trees showed that in the wounded areas, the cambium and vascular tissue regenerate very fast both *in vivo* [19, 20] and *in vitro* [25, 65, 66]. Regeneration is accompanied by numerous anticlinal divisions of cambial cells and their dynamic intrusive growth [19, 20, 64], which finally leads to the reconstruction of vasculature and new vessel patterning in the incised regions [25, 65]. In some instances, when the auxin flow is locally reversed, the so-called circular vessels develop [32, 67, 68]. In the nondisturbed woody plants, circular vessels are often found in branch junctions, above the axillary buds [68], whereas in incised plants, after transversal cuts and exogenous auxin application to stem segments, in wounded regions [32, 67]. Accordingly, circular vessels occur in the form of rings and are presumably induced as a consequence of the circular auxin flow and the establishment of the circular polarity of individual cells that dedifferentiated into this type of vessels [67]. Thus, according to Sachs and Cohen [67], circular vessels develop as a response of individual cells to the auxin flux rather than to the high local auxin concentration. In nonwoody dicotyledonous plants characterized by primary tissue architecture, such as *Phaseolus vulgaris*, *Pisum sativum*, or *Coleus* sp., vasculature is regenerated directly from dedifferentiated parenchyma cells [1–5]. New vessels are arranged either around the wound according to the presumable new auxin flow [69] or form the socalled bypass strands directly through the wound [3] or bridges between the neighboring vascular bundles [70]. Lack of the vascular cambium in the studied nonwoody plants restricted a detailed analysis of regeneration of this meristematic tissue and cellular events accompanying this process. Therefore in the used models, the most intriguing questions are still remained of

Patterning of vascular tissue and variety of tracheary elements developed as a dynamically operating water-conducting system and was extensively studied in the woody plants [13, 14]. However, mechanism regulating xylogenesis at cellular and molecular levels remains unclear, and many questions are unanswered. For example, differentiation of tracheids as a type of tracheary elements commonly found in trees, but for the first time detected in mechanically stimulated *Arabidopsis*, led to important conclusions about the involvement of the artificial weight in wood formation. Following stages of xylogenesis involving formation of the variety of tracheary elements, such as recognized tracheids, will be helpful in future

secondary xylem vessels; *vc*, vascular cambium; *co*, cortex); bars, 50 μm (A); 20 μm (C).

**Figure 4.** Secondary xylem in the weight stimulated stems of *Arabidopsis*. (A) Secondary xylem elements, like vessels and fibers, are produced from cambial derivatives after numerous periclinal divisions of fusiform cambial cells. Cortex parenchyma is visible outside the secondary vascular tissues. (B) Schematic visualization of the tissue arrangement in stem and localization of the tissues showed in (A) is indicated by the square. (C) Vessel strand developed parallel to longitudinal axis of stem. Characteristic patterning of the secondary cell wall (arrow) and perforation plates developed on the opposite apical-basal ends of neighboring vessel members (circle) determines the most diagnostic features for this type of tracheary elements (A and C, bright-field images in a confocal laser-scanning microscope; *fb*, fibers; *sxv*,

analysis.

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answer: (1) what is the role of vascular cambium in vascular tissue regeneration?, (2) which of the cellular events are temporary correlated with the vascular cambium regeneration?, (3) is vascular cambium regeneration mediated by the canalization process? Full verification of the postulated canalization hypothesis and identification of the molecular mechanisms accompanied vascular tissue regeneration are still limited.

Because of the difficulties in using woody plants as a convenient model system [52], mechanisms of cambium regeneration are still poorly understood. With the *Arabidopsis* "cambium" model, it is now possible to monitor vascular tissue regeneration with all cellular events accompanying this process. Thus, in control conditions, i.e., in nonincised stems, polar auxin flow is in the direction from apical to basal part of stems, and according to this flow, new vasculature develops. Otherwise, in incised stems (i.e., wounded stems), polar auxin transport is disturbed; thus, new ways of auxin flow are established. As a consequence, new vessel strand arrangement is changed, because the new vasculature likely developed according to new directions of auxin cell-to-cell transport (**Figure 5**). In wounded *Arabidopsis* stems, threads of new vessel strands develop above or around a wound (**Figure 5A** and **B**, respectively). Interestingly, vessels above a wound regenerated faster, in the first days after wounding (DAW) (2 and 3 days), whereas vessel around a wound differentiated in the next few days, beginning the day 4 and circumventing the incised areas. They developed from cells after their numerous, uneven divisions,

**Figure 5.** Paths of vessel regeneration in wounded *Arabidopsis* stems. (A) Threads of short vessel members developed above a wound. (B) Vessel strands regenerated around a wound. (C) Vessel "bypass" strands reconstructed partially from the callus tissue developed inside the wound. Arrows indicate regenerated vessel strands. Broken arrows mark places of the wound; bright-field images in a confocal laser-scanning microscope; bars, 50 μm.

what is commonly observed in the wounded tissue. Differentiating vessels were visualized by the activity of the *AtHB8* gene, which belongs to HD-ZIP III family [71, 72]. The *AtHB8* is positively regulated by auxin, and its extensive activity in wounded regions during vascular tissue regeneration suggested that *AtHB8* might play a crucial role in the vasculature development [71, 72]. The last observed way of vasculature regeneration is correlated with callus differentiation (**Figure 5C**). Namely, in wounded areas vessels develop from previously proliferated callus tissue cells. Such vessels often create the type of "bypass" strands extending above and below the transversal incision.

answer: (1) what is the role of vascular cambium in vascular tissue regeneration?, (2) which of the cellular events are temporary correlated with the vascular cambium regeneration?, (3) is vascular cambium regeneration mediated by the canalization process? Full verification of the postulated canalization hypothesis and identification of the molecular mechanisms accompa-

Because of the difficulties in using woody plants as a convenient model system [52], mechanisms of cambium regeneration are still poorly understood. With the *Arabidopsis* "cambium" model, it is now possible to monitor vascular tissue regeneration with all cellular events accompanying this process. Thus, in control conditions, i.e., in nonincised stems, polar auxin flow is in the direction from apical to basal part of stems, and according to this flow, new vasculature develops. Otherwise, in incised stems (i.e., wounded stems), polar auxin transport is disturbed; thus, new ways of auxin flow are established. As a consequence, new vessel strand arrangement is changed, because the new vasculature likely developed according to new directions of auxin cell-to-cell transport (**Figure 5**). In wounded *Arabidopsis* stems, threads of new vessel strands develop above or around a wound (**Figure 5A** and **B**, respectively). Interestingly, vessels above a wound regenerated faster, in the first days after wounding (DAW) (2 and 3 days), whereas vessel around a wound differentiated in the next few days, beginning the day 4 and circumventing the incised areas. They developed from cells after their numerous, uneven divisions,

**Figure 5.** Paths of vessel regeneration in wounded *Arabidopsis* stems. (A) Threads of short vessel members developed above a wound. (B) Vessel strands regenerated around a wound. (C) Vessel "bypass" strands reconstructed partially from the callus tissue developed inside the wound. Arrows indicate regenerated vessel strands. Broken arrows mark

places of the wound; bright-field images in a confocal laser-scanning microscope; bars, 50 μm.

nied vascular tissue regeneration are still limited.

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Regeneration of vascular tissue in wounded *Arabidopsis* stems is accompanied by temporal and spatial changes following new vessel development. New vessel strands regenerated in the incised regions around a wound develop as a consequence of cambial cell regeneration. Longitudinal continuum of vascular cambium is disturbed after the transversal cut. In such experimental system, rapid auxin response is found as a primary signal of the regeneration. Merely at the first day after incision, elevated auxin concentration is observed above a wound and in the next few days also around a wound [8]. Vasculature regeneration is strictly correlated with tissue repolarization and establishment of new polarity in neighborhood of the wound. Tissue repolarization always preceded emergence of PIN1-positive auxin channels (**Figure 6**). As a consequence, layer of new vessels develops around a wound, and the regenerated vasculature becomes enlarged

**Figure 6.** Schematic visualization of the temporal and spatial changes during vascular tissue regeneration in following days after wounding (DAW): re-localization of PIN1 in cells around a wound (1 DAW); establishment of new position of PIN1 at cellular plasma membranes (2 DAW); increased auxin concentration in auxin channels (3 DAW); expression of *AtHB8* gene in differentiating vessels (4 DAW).

in the days following the incision. Analysis of regeneration process in incised *Arabidopsis* stems strongly supported canalization hypothesis. Emergence of new vasculature is correlated here with elevated auxin response and changed polarity in auxin channels, from which new vessel strands develop in the wounded areas.
