**5. Genetic control of vascularization processes**

early embryogenesis or plant organ initiation, are strictly correlated with the establishment of local PIN-dependent auxin gradients that precede cell divisions and differentiation [54, 55, 87]. The expression of auxin efflux carrier genes, like *PIN1*, *PIN2*, *PIN3*, *PIN4*, and *PIN7* was found to peak at the inflorescence stems of *Arabidopsis* during their maturation and secondary vascular tissue development. Changes in PIN localization and tissue polarity in response to auxin that are presumably related to the directional vascular tissue patterning have been observed and modeled [4, 5, 46, 88]. Moreover, in wounded pea or bean epicotyls, the PIN polarity was gradually rearranged marking the position of differentiating vessel strands [4, 5]. Emergence of auxin channels is here visualized by *PIN1* expression of the cellular auxin transporters. In *Arabidopsis* model with mechanically stimulated inflorescence stems, the subcellular PIN1 position was gradually stabilized and restricted only to cell sides in a first few days after weight application, along the presumable direction of the auxin flow [8]. The auxin-dependent canalization is strongly supported by studies on leaf vein patterning and on the role of the genes encoding the auxin response factor *MONOPTEROS* (*MP*) and *PIN1* [47]. Dynamic expression of both of the genes and gradual establishment of polarized PIN1 protein localization indicates the direction auxin flow during the vascular tissue patterning in analyzed leaves [47]. Moreover, the other *Arabidopsis* gene *GNOM/ EMB30*, which affects apical-basal position of PIN1, seems to be required for regulation of

The role of auxin transporters in vascular tissue patterning is clearly visible in wounded inflorescence stems of *Arabidopsis*, during vascular cambium regeneration [8]. Rapid tissue repolarization indicated by reposition of PIN1 at cellular plasma membranes of differentiating cells is emphasized. Dynamic temporal changes in tissue polarity are correlated with varied auxin response and its accumulation above and around a wound. Whereas auxin concentration arises in few hours after wounding, maximum of auxin levels is established at auxin channels and preceded establishment of new polarity in wounded areas of *Arabidopsis* stems. Cellular auxin transporters are characterized with changed position of PIN1 proteins. Thus, direction of auxin flow through the auxin channels is precisely determined. Both of the events are strictly correlated with each other and play a decisive role in

Two related protein families—Aux/IAA and ARFs—are well-known key regulators of auxinmodulated gene expression and act in the TIR1-mediated signaling pathway [89, 90]. Members of ARF family share the characteristic arrangement of a highly conserved DNA-binding domain near the N-terminus, which appear to be capable to auxin response elements (AuxREs)—short conserved sequences (TGTCTC) that have been shown to be essential for auxin regulation of auxin-inducible genes [6]. It is likely that the ARF proteins are strongly involved in the vascularization downstream proteolytic SCFTIR1 complex machinery [80]. In support of this, an increased level of ARF transcripts was differentially regulated during the secondary growth, and three of them (ARF2, ARF4, and ARF5) had the most dramatic expression changes, indicating their putative roles in apical-basal signaling and xylogenesis [6, 42]. On the other hand, the *AUXIN-RESISTANT 1* (*AXR1*) gene is required for normal *TIR1* function and, when mutated,

the coordinated tissue polarity [6].

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vascular tissue development.

**4.3. Auxin signaling pathways in vascular tissue patterning**

Numerous genes differentially regulated during vascularization in woody plants are expected to be identified with the use of obtained *Arabidopsis* model. Many of the genes were indicated to be involved in auxin responses implicating auxin engagement in regulation of vascular tissue development and patterning [42, 43], and the same is expected for vascular tissue regeneration. Experimental data have proven a key role of auxin in variety of developmental processes [80]; however, the molecular, auxin-mediated mechanism involved in vasculature regeneration remains mostly unknown.

As indicated recently, transcription factor genes promoting secondary growth induction [42, 43] can be applied in genetic transformation to improve our knowledge on xylogenesis and regeneration capacity of woody plants. Several regulatory genes, like *NAC*-domain genes or key regulators of SAM, shoot apical meristem, organization (*WUS, STM*, *WOX*, *CLV*s), are closely associated with the vascular tissue formation. It was reported that two *NAC*-genes, *NAM* and *CUC*, and *ANT*—a cell proliferation marker—play potential roles in vascular tissue differentiation and function [96]. *ANT* regulates organ growth through the maintenance of meristematic tissue activity.

The expression of the homeobox genes (*AtHB*s) is highly increased during xylem production and regarded as a positive regulator of the activity of procambial and cambial cells to differentiate [71, 72]. Baima et al. [72] reported ectopic expression of *AtHB-8* gene during vascular regeneration after wounding in *Arabidopsis*. Extensive activity of this gene was found in the regenerated tissues, suggesting intensive transcriptional reprogramming during new vessel development. In the model with functioning vascular cambium, expression of *AtHB-8* is observed in differentiating cambial derivatives, in early stages of their maturation into the vessels (**Figure 8**).

Homeostasis of vascular cambium with its non-disturbed functionality plays an important role in the vascularization [59]. However, genetic control of vascular cambium activity is poorly characterized. The role of the leucine-rich repeat receptor-like kinase (LRR-RLK) families in regulation of this lateral meristem homeostasis, underlying the role of *CLV1* (*CLAVATA1*) well-known apical meristem marker and PXY (phloem intercalated with xylem)—a cambiumspecific receptor-like kinase, in this process is suspected [59]. Two receptor-like kinases MOL1 (more lateral growth1) and RUL1 (reduced in lateral growth1) identified as opposing regulators of cambium activity were also reported [59]. Recently, the role of the homeobox transcription factor WOX4 (wuschel-related homeobox 4), as an essential cambium regulator positively regulated by *PXY*, has been revealed [59, 97]. Since many years the correlations between the cytoskeleton dynamic (both actin filaments and microtubules) and activity and functioning of vascular cambium [31, 37, 98] have been widely discussed. It was postulated that changes in

**Figure 8.** The *AtHB8* gene expression in differentiating vessels. Longitudinal tangential section through the basal part of stem. Expression of *AtHB8* in differentiating vessels (arrows); asterisk indicates maturated vessel (*AtHB8:GUS* transgenic line; LR white resin section; bar: 20 μm).

microtubule orientation might be specific cellular marker for cambial cells in the stage of their differentiation into tracheary elements. Analyses of the following steps of this process revealed the dynamic changes in the microtubule orientation in the differentiating cambial cells [98].

tissues, suggesting intensive transcriptional reprogramming during new vessel development. In the model with functioning vascular cambium, expression of *AtHB-8* is observed in differentiating cambial derivatives, in early stages of their maturation into the vessels (**Figure 8**).

Homeostasis of vascular cambium with its non-disturbed functionality plays an important role in the vascularization [59]. However, genetic control of vascular cambium activity is poorly characterized. The role of the leucine-rich repeat receptor-like kinase (LRR-RLK) families in regulation of this lateral meristem homeostasis, underlying the role of *CLV1* (*CLAVATA1*) well-known apical meristem marker and PXY (phloem intercalated with xylem)—a cambiumspecific receptor-like kinase, in this process is suspected [59]. Two receptor-like kinases MOL1 (more lateral growth1) and RUL1 (reduced in lateral growth1) identified as opposing regulators of cambium activity were also reported [59]. Recently, the role of the homeobox transcription factor WOX4 (wuschel-related homeobox 4), as an essential cambium regulator positively regulated by *PXY*, has been revealed [59, 97]. Since many years the correlations between the cytoskeleton dynamic (both actin filaments and microtubules) and activity and functioning of vascular cambium [31, 37, 98] have been widely discussed. It was postulated that changes in

**Figure 8.** The *AtHB8* gene expression in differentiating vessels. Longitudinal tangential section through the basal part of stem. Expression of *AtHB8* in differentiating vessels (arrows); asterisk indicates maturated vessel (*AtHB8:GUS* transgenic

line; LR white resin section; bar: 20 μm).

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The most important role in the whole vascular-formation and regeneration machinery is played by auxin response genes referred to as early/primary auxin response genes. Three major classes of these genes, *Aux/IAA*, *SAURs*, and *GH3*, are characterized by the so-called auxin response elements (AuxREs), the TGTCTC-containing auxin response promoter elements. The genes are specifically induced by auxin, which may rapidly regulate their transcription. Accordingly, when auxin concentration in cells is low, auxin response genes are repressed. Oppositely, when auxin concentration is elevated, transcription of the genes is activated in a few minutes [99], and ARF transcription factors are binding to its AuxRE target sides. It is discussed that dependently on the tissue and developmental stages of the plants, tissue-specific expression of both ARF and Aux/IAA proteins could regulate the transcription of different sets of genes for different developmental processes.

Studies in the *Populus tomentosa* [29] have shown that different groups of genes are activated dependent on the stage of the secondary vascular tissue development. Thus, in the cambial zone, high expression of the *ARK1* gene was observed. It was suggested that *ARK1* regulates cambium activity including cambial cell divisions and differentiation of the cambial derivatives [30]. On the contrary, during the secondary xylem formation, expression of the group of genes responsible for the tracheary element differentiation and maturation, such as *SND1* or *NST1* and *NST3*, was reported [100, 101].

It is widely postulated that important role in the vascularization is played also by gene expression that accompanies this process, like *PLT* (*PLETHORA*) or *TCH* (*TOUCH*) genes encoding calcium-binding proteins. Whereas *PLT*s regulate de novo shoot regeneration in *Arabidopsis* by controlling successive developmental events, *TCH* genes (*TCH2* and *TCH4*) strongly induced by mechanical stimuli like touch and wind [42] may be involved in the signal transduction and secondary xylem formation. Otherwise, two of the *VASCULAR-RELATED NAC-DOMAIN* genes (*VND6* and *VND7*) are reported as positive xylem vessel differentiation regulators in both *Arabidopsis* and poplar [48]. Thus, the elusive mechanism for auxin-regulated vascular tissue patterning might be a part of the extensive genetic network including several hormonal signaling pathways and dynamic spatiotemporal switched on/ off gene expression. Deciphering of these yet unknown relationships will help to translate the mechanisms regulated in vascular tissue development and regeneration in woody plants.
