**4. Role of plant hormone auxin and auxin transporters in vascular tissue development**

Auxin is regarded as a multifunction plant hormone, which plays a fundamental role in developmental processes during organo- and morphogenesis. Auxin is a primary signal in regulation of many cellular processes, which control oriented divisions, cell elongation, or differentiation. At last, auxin is a key hormonal factor inducing vascularization—vascular tissue development, patterning, and regeneration. Polar auxin transport (PAT) manifested as physiological, basipetal direction of auxin flow represents a unique mechanism specific to plants. The cellular and molecular action of this process, explained in the chemiosmotic model, is based on auxin influx and efflux carriers, namely, AUX and PIN proteins, which actively participate in the cell-to-cell hormone transport [73–75]. The local auxin accumulation, its minima and maxima, or the so-called gradients in tissues are precisely controlled by this process.

#### **4.1. Auxin as a primary signal inducing vascularization**

The role of auxin as a primary signaling cue in vascularization has been widely discussed for decades. Experiments with radioactively labeled auxin show its maximum concentration in the meristematic tissues such as cambium [22, 57] and in adjacent cambial derivatives, differentiating into xylem [76]. Periodic fluctuation of auxin concentration in cambium influences the frequency of cambial cell divisions, production of cambial derivatives, and secondary vascular tissues. Disturbance of these correlations leads to many defects in cambium functioning and xylem formation. Using transgenic lines of *Arabidopsis*, elevated auxin response is easily found just in the cambial cells of both types of cambia (**Figure 7**). Auxin concentration is very high in the fascicular cambium bands, primary meristematic tissue in the vascular bundles (**Figure 7**), as well as in the interfascicular vascular cambium on the stem circumference (**Figure 7**).

From the experimental studies on the vascularization *in vitro*, it appears that parenchyma callus tissue is the most convenient for the analysis. Previously uniform callus can form vascular tissue bands or groups of vessels differentiation. However, the process can be realized only in the sufficiently thick callus tissue. It is shown that differentiated xylem in surrounded by cambium-like cells, which additionally are able to produce phloem elements in the inner callus regions. Auxin-dependent vascularization is also shown in the studies with young *Syringa* sp. stems [77]. Combination of auxin and sucrose decides about the induction of vascularization in the axillary buds *in vitro*. Moreover, dependent on the hormone and sucrose concentration, varied vascular tissues develop.

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

**4. Role of plant hormone auxin and auxin transporters in vascular tissue** 

Auxin is regarded as a multifunction plant hormone, which plays a fundamental role in developmental processes during organo- and morphogenesis. Auxin is a primary signal in regulation of many cellular processes, which control oriented divisions, cell elongation, or differentiation. At last, auxin is a key hormonal factor inducing vascularization—vascular tissue development, patterning, and regeneration. Polar auxin transport (PAT) manifested as physiological, basipetal direction of auxin flow represents a unique mechanism specific to plants. The cellular and molecular action of this process, explained in the chemiosmotic model, is based on auxin influx and efflux carriers, namely, AUX and PIN proteins, which actively participate in the cell-to-cell hormone transport [73–75]. The local auxin accumulation, its minima and maxima, or the so-called gradients in tissues are precisely controlled

The role of auxin as a primary signaling cue in vascularization has been widely discussed for decades. Experiments with radioactively labeled auxin show its maximum concentration in the meristematic tissues such as cambium [22, 57] and in adjacent cambial derivatives, differentiating into xylem [76]. Periodic fluctuation of auxin concentration in cambium influences the frequency of cambial cell divisions, production of cambial derivatives, and secondary vascular tissues. Disturbance of these correlations leads to many defects in cambium functioning and xylem formation. Using transgenic lines of *Arabidopsis*, elevated auxin response is easily found just in the cambial cells of both types of cambia (**Figure 7**). Auxin concentration is very high in the fascicular cambium bands, primary meristematic tissue in the vascular bundles (**Figure 7**), as well as in the interfascicular vascular cambium on the stem circumfer-

From the experimental studies on the vascularization *in vitro*, it appears that parenchyma callus tissue is the most convenient for the analysis. Previously uniform callus can form vascular tissue bands or groups of vessels differentiation. However, the process can be realized only in the sufficiently thick callus tissue. It is shown that differentiated xylem in surrounded by cambium-like cells, which additionally are able to produce phloem elements in the inner callus regions. Auxin-dependent vascularization is also shown in the studies with young *Syringa* sp. stems [77]. Combination of auxin and sucrose decides about the induction of vascularization in the axillary buds *in vitro*. Moreover, dependent on the hormone and sucrose concentration,

strands develop in the wounded areas.

**4.1. Auxin as a primary signal inducing vascularization**

**development**

126 Plant Engineering

by this process.

ence (**Figure 7**).

varied vascular tissues develop.

**Figure 7.** Elevated auxin concentration in cambium of non-incised *Arabidopsis* control stems. Increased auxin level in fascicular cambium (vascular bundle, asterisk) and in the interfascicular cambial cells (double asterisks); cross section through the basal part of Col-0 stem; confocal laser-scanning microscope; IAA immunolocalization-staining with the polyclonal anti-IAA antibody (white color in cambial cells; dilution 1:500); bar, 50 μm.

Several reports discussed auxin as a specific morphogenetic signal triggering cell fates during vascular tissue development and its maturation [78]. Locally created centers characterized by elevated auxin response become more competent for auxin flow through primarily uniform tissues. Auxin waves created in plant organs as a specific system of hormonal information that decide about realization of many developmental programs in plants, among them cambial activity and differential cambial responses [79, 80]. Thus analogically, gradual emergence of auxin channels and gradually narrowing auxin flow finally results in vascular strand differentiation. In other words, canalized auxin flux determined the paths of new vasculature development.

The canalization-predicted vasculature formation is especially observed during regeneration process, in new regenerated vessels after incision [1, 2, 4, 5, 8, 81]. Particularly important contributions to the role of auxin in the vascular tissue differentiation brought studies on *Pisum* sp. [1, 2]. According to all experiments performed by Sachs, vascularization depends on the polar auxin transport, and new vascular band induction depends on the auxin concentration and polarity. Moreover, the early stages of vascular band differentiation are related to the canalization of the polar auxin flow. A key role of auxin in promotion of canalized flow by itself and transport channels formation is commonly accented. However, the feedback mechanism between auxin flow, polarity, and vessel formation as a response to concentration gradients or directional auxin fluxes remains unclear [82, 83].

#### **4.2. Role of auxin transporters in cellular and tissue polarity**

The positive feedback loop between polar auxin flow and the polar, subcellular localization of the PIN-FORMED (PIN) auxin transport proteins [56] that, in turn, determine the auxin flow directionality is widely studied [53, 54, 84–86]. Many developmental processes, such as 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 coordinated tissue polarity [6].

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 vascular tissue development.

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

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, changes the stabilization dynamics of the Aux/IAA proteins [71]. Mutations in the TIR1/AFBs make Aux/IAA proteins insensitive to auxin and can therefore keep ARF transcription factors and auxin signaling repressed. Thus, ARFs together with Aux/IAA proteins constitute a central mechanism in auxin signaling during plant development [91].

Auxin besides regulating a gene expression by the TIR1/AFB pathway can also inhibit internalization of the PIN proteins by a feedback regulation [92]. The underlying perception and signaling mechanism is unclear, but it does not involve transcription regulation and is distinct from the TIR pathway [93]. It may relay on the Auxin Binding Protein 1 (ABP1) since the ABP1 overexpressors increase the PIN internalization and mutations in the auxin binding pocket of ABP1 make the ABP1 effect on PIN internalization auxin-insensitive [88]; however, due to unreliable loss-of-function data [94, 95], this issue requires further clarification.

The identification of spatiotemporal gene expression pattern and the key components of auxin signaling pathway/pathways will greatly contribute to understanding of the molecular mechanisms involved in auxin-induced regeneration switch in cambial cells. In addition, the knowledge on genetic factors, such as ARFs, AFBs involved in the SCFTIR1 auxin receptor complex, PIN auxin efflux transporters, or AtHB family of early vascularization markers determining developmental plasticity of cambial cells, can be useful in genetic improvement of woody plants for environment and biotechnology purposes.
