**2. Secondary vascular tissues in woody plants**

Vascular tissue is a well function conducting system typical for all woody plants, among them in trees. In young plants, characteristic primary tissues such as procambium, primary xylem, and primary phloem, develop. During the secondary growth, vascular tissue undergoes the transition from primary into the secondary vascular patterning. Vascular cambium, secondary xylem, and secondary phloem form a closed ring on the stem circumference. They are arranged in the radial rows as a consequence of periclinal divisions of cambial cells [10–12]. The secondary growth is mostly characteristic for all woody plants, and production of the secondary vascular tissues is an important developmental feature of the plants [12, 13].

#### **2.1. Vascular cambium**

Vascular cambium plays a crucial role in the secondary growth and vascular tissue patterning in woody plants [14, 15]. Activity and functioning of vascular cambium decide about the amount of the secondary phloem and xylem, which are produced outward and inward the vascular cambium, respectively [12, 13]. This meristematic tissue is built from two types of cells: ray cambial cells producing secondary rays—transverse conducting system in tissues—and fusiform cambial cells, producing elements of the longitudinal conducting systems in woody plants. Characteristic feature of the vascular cambium is intrusive growth of the fusiform cambial cells and their periclinal divisions [13, 16, 17]. The intrusive growth is restricted to the ends of growing cells, when two neighboring cells grow in opposite directions. Periclinal divisions of cambial cells decide about production of cambial derivatives and secondary tissue element differentiation.

Vascular cambium is the tissue very much sensitive to mechanical injuries, such as wounding or grafting. However, it can easily regenerate under suitable conditions. It has been experimentally shown that cambium regeneration is mostly dependent on the tensile stress and pressure. Results obtained by Brown [18] indicate that cambium activity, cell divisions, and xylem formation can be easily affected by the pressure externally implied to the cambial strips. It is also documented in *in vivo* experiments with the wounding stems of *Larix europea* that regeneration of this meristematic tissue can dynamically progress under the tensile stress and pressure implication. In such cases, even low pressure (25 kPa) implied to cambium in wounded areas decides about its very rapid regeneration. Lack of the mechanical factors leads to abundant callus tissue production [19, 20].

It has been postulated that appropriate functioning of vascular cambium and its cyclic activity, i.e., periclinal divisions during the seasons, is strictly correlated with auxin signaling and auxin responses [21–23]. From the studies on *Populus tremula* L. × *Populus tremuloides*, it appears that the highest auxin concentration is found in the layer of cambium. Auxin plays here a key role in the regulation of cambial cell divisions and elongation [23]. According to the results obtained with the vascular cambium in *Pinus sylvestris*, thickness of the cambial layer is directly dependent on the auxin concentration in the tissue, which stimulates frequency of divisions [21, 22]. The highest activity of vascular cambium is found at the beginning of the vegetative seasons, in early spring, which is correlated with the first periclinal divisions of the cambial cells, production of new cambial derivatives, and their differentiation into new secondary vascular tissues. Otherwise, the lowest cambial activity in winter, during the dormant period, is strictly correlated with decreasing both temperature and hormonal levels in cambium layer [24]. Periclinal divisions are limited and almost completely stop; thus, the vasculature is not produced during this time. However, it was experimentally shown that such situation could be easily reversed after exogenous auxin application [25]. As a consequence, activity of cambium and periclinal divisions of cambial cells can be resumed by auxin. Thus, from all the studies on the woody plants, it appears that elevated auxin response in cambial cells as well as fluctuations of auxin (maxima/minima) in cambium plays decisive role in seasonal nature of the trees, for example, switching on/off dormant periods [21, 22]. Changes of the cell wall components [26–28] and gene expression during the cyclic activity of vascular cambium [29, 30], correlated with the rapid cytoskeleton rearrangement in differentiating cells [31], indicate that this meristematic tissue plays a crucial role in the secondary growth of woody plants and decides about their adaptation to variable environmental conditions.

#### **2.2. Secondary xylem**

In this review, we summarize information concerning secondary vascular tissue development in *Arabidopsis* including cambium ontogenesis and xylogenesis, with accompanying changes in auxin distribution, directionality of its flow, and cellular polarity defined by auxin transporters (PIN family proteins), which have been indicated to be involved in regulation of vas-

Vascular tissue is a well function conducting system typical for all woody plants, among them in trees. In young plants, characteristic primary tissues such as procambium, primary xylem, and primary phloem, develop. During the secondary growth, vascular tissue undergoes the transition from primary into the secondary vascular patterning. Vascular cambium, secondary xylem, and secondary phloem form a closed ring on the stem circumference. They are arranged in the radial rows as a consequence of periclinal divisions of cambial cells [10–12]. The secondary growth is mostly characteristic for all woody plants, and production of the secondary vascular tissues is an important developmental feature of

Vascular cambium plays a crucial role in the secondary growth and vascular tissue patterning in woody plants [14, 15]. Activity and functioning of vascular cambium decide about the amount of the secondary phloem and xylem, which are produced outward and inward the vascular cambium, respectively [12, 13]. This meristematic tissue is built from two types of cells: ray cambial cells producing secondary rays—transverse conducting system in tissues—and fusiform cambial cells, producing elements of the longitudinal conducting systems in woody plants. Characteristic feature of the vascular cambium is intrusive growth of the fusiform cambial cells and their periclinal divisions [13, 16, 17]. The intrusive growth is restricted to the ends of growing cells, when two neighboring cells grow in opposite directions. Periclinal divisions of cambial cells decide about production of cambial derivatives and

Vascular cambium is the tissue very much sensitive to mechanical injuries, such as wounding or grafting. However, it can easily regenerate under suitable conditions. It has been experimentally shown that cambium regeneration is mostly dependent on the tensile stress and pressure. Results obtained by Brown [18] indicate that cambium activity, cell divisions, and xylem formation can be easily affected by the pressure externally implied to the cambial strips. It is also documented in *in vivo* experiments with the wounding stems of *Larix europea* that regeneration of this meristematic tissue can dynamically progress under the tensile stress and pressure implication. In such cases, even low pressure (25 kPa) implied to cambium in wounded areas decides about its very rapid regeneration. Lack of the mechanical factors leads

cular tissue patterning and regeneration [7–9].

the plants [12, 13].

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**2.1. Vascular cambium**

secondary tissue element differentiation.

to abundant callus tissue production [19, 20].

**2. Secondary vascular tissues in woody plants**

In the most typical form, secondary xylem, also called a wood, is found in stems and roots of the woody plants. The secondary xylem, a longitudinal conducting system in trees, develops from the cambial derivatives, which during the maturation process is differentiated into elements of the wood-like vessels, fibers, and tracheids [13, 15].

Vessels of the secondary xylem form strands parallel to the longitudinal axis of the organs stems or roots. Every vessel strand is consisted of single vessel elements, the so-called vessel members, connected with each other by open perforation plates localized on their apical-basal ends [12]. It is postulated that direction of vessel differentiation is dependent on direction of auxin flow. Thus, in nondisturbed stems, vessels developed according to the polar auxin transport (PAT) in the apical-basal direction whereas in incised organs according to newly established direction of auxin flow—circumventing the wounded regions. Correlations between auxin flow and vasculature patterning were experimentally documented in woody plants after wounding [32] as well as nonwoody models [4, 5, 8]. Characteristic feature for all types of vessels (primary protoxylem, metaxylem, and secondary xylem vessels) is the secondary cell wall. Different patterning of the secondary cell wall is realized during vessel maturation process and depends on the type of vessel [14]. During the maturation process, protoplasts of differentiating vessels disappeared. Frequently, the lumen of the vessel members is enlarged in comparison to other tracheary elements, mainly in a wood of such species as *Fraxinus excelsior*, *Quercus borealis*, or *Ulmus americana* [13]. In many cases, length of the vessel members is different than the length of the fusiform cambial cells from which they developed. Interestingly, longitudinal vessel strands change the orientation to the longitudinal axis of stems. Such fluctuations are observed as the wavy grain patterning of wood in many trees [33].

Fibers of the secondary xylem are recognized as one of the longest tracheary elements, characterized by the tapered cell ends and reduced lumen. As a consequence of their intensive intrusive growth, fibers could be even few times longer than the fusiform cambial cells and their derivatives. Particular type of the woody fibers is the so-called gelatin fibers, developed as a layer of the reaction wood in many deciduous as well as coniferous trees, i.e., *Populus* sp. or *Picea* sp. Inner layer of secondary cell wall of these fibers is built mainly from cellulose. The presence of the callose and callose-like cell wall components plays here an important role in mechanical properties of the wood [34].

Tracheids, other tracheary elements of secondary xylem, are nonperforated, long cells with the bordered pits. Dependently on the type of a wood, tracheids are classified as (1) vessel-like tracheids arranged in longitudinal, similar to vessels conducting strands, commonly found in *Carpinus* sp., *Ulmus* sp., *Acer* sp., or *Tilia* sp.; (2) tracheids differentiated around the vessels with enlarge lumen, adjacent to them, and strictly surrounding; and (3) fiber-like tracheids [35]. The last of them develop as a conducting and storage water system but also play mechanical functions and in some species could be the main component of the softwood of gymnosperms [36]; they are found also in some angiosperms, i.e., in *Populus* sp.

Besides of the dead, water-conducting elements of the secondary xylem mentioned above, in many cases secondary vascular tissue of woody plants is compound with the xylem parenchyma cells, which remain alive for a long time to finally die in the programmed cell death (PCD) process [12].

Thorough knowledge about the genetic and molecular mechanisms involved in vascular tissue functioning, development, and regeneration is eagerly expected. Different molecular components involved in the determination of developmental plasticity of cambial cells have been searched for with special interest focused on the key regulators of vascularization. Genes involved in auxin response, auxin signaling pathways, and tissue and cellular polarity during vascular tissue development induced in vascular cambium should be extensively studied for detailed characterization of this process.

#### **2.3.** *Arabidopsis* **as a nonwoody plant example for vascular tissue formation**

Since many years, *Arabidopsis* is nominated as a good model for studies of vascular tissue formation, because under suitable conditions, *Arabidopsis* can undergo secondary growth in hypocotyls, when enlarged layer of secondary xylem develops during xylogenesis [37]. Xylogenesis in hypocotyls is comparable to xylogenesis in roots. Development of secondary xylem is divided here into two phases: the early phase, xylem is building from vessels and numerous parenchyma cells, and in the second, later phase, also called xylem expansion, enlarged amount of xylem elements develops mainly vessels and fibers [37, 38]. It is well documented that vascular tissue develops not only in hypocotyls [37] but also in the matured inflorescence stems [39–41], in their basal parts [42–45]. With the use of *Arabidopsis*, the correlation between auxin signaling and tissue polarity has been intensively studied and modeled [46–49]. However, because of lack of some important vasculature features such as a variety of typical phenotype features and functional cambium, these models could not be used for full analysis and description of vascular tissue development and compared to the analogical process in trees. For example, in both *Arabidopsis* models mentioned above, rays were not found. Also intrusive growth typical for fusiform cambial cells was not confirmed. Finally, impressive variety of tracheary elements, among them tracheids, commonly found in trees, were not observed in *Arabidopsis* mature stems and hypocotyls, which underwent secondary growth [50].

lumen of the vessel members is enlarged in comparison to other tracheary elements, mainly in a wood of such species as *Fraxinus excelsior*, *Quercus borealis*, or *Ulmus americana* [13]. In many cases, length of the vessel members is different than the length of the fusiform cambial cells from which they developed. Interestingly, longitudinal vessel strands change the orientation to the longitudinal axis of stems. Such fluctuations are observed as the wavy

Fibers of the secondary xylem are recognized as one of the longest tracheary elements, characterized by the tapered cell ends and reduced lumen. As a consequence of their intensive intrusive growth, fibers could be even few times longer than the fusiform cambial cells and their derivatives. Particular type of the woody fibers is the so-called gelatin fibers, developed as a layer of the reaction wood in many deciduous as well as coniferous trees, i.e., *Populus* sp. or *Picea* sp. Inner layer of secondary cell wall of these fibers is built mainly from cellulose. The presence of the callose and callose-like cell wall components plays here an important role in

Tracheids, other tracheary elements of secondary xylem, are nonperforated, long cells with the bordered pits. Dependently on the type of a wood, tracheids are classified as (1) vessel-like tracheids arranged in longitudinal, similar to vessels conducting strands, commonly found in *Carpinus* sp., *Ulmus* sp., *Acer* sp., or *Tilia* sp.; (2) tracheids differentiated around the vessels with enlarge lumen, adjacent to them, and strictly surrounding; and (3) fiber-like tracheids [35]. The last of them develop as a conducting and storage water system but also play mechanical functions and in some species could be the main component of the softwood of

Besides of the dead, water-conducting elements of the secondary xylem mentioned above, in many cases secondary vascular tissue of woody plants is compound with the xylem parenchyma cells, which remain alive for a long time to finally die in the programmed cell death

Thorough knowledge about the genetic and molecular mechanisms involved in vascular tissue functioning, development, and regeneration is eagerly expected. Different molecular components involved in the determination of developmental plasticity of cambial cells have been searched for with special interest focused on the key regulators of vascularization. Genes involved in auxin response, auxin signaling pathways, and tissue and cellular polarity during vascular tissue development induced in vascular cambium should be extensively studied for

Since many years, *Arabidopsis* is nominated as a good model for studies of vascular tissue formation, because under suitable conditions, *Arabidopsis* can undergo secondary growth in hypocotyls, when enlarged layer of secondary xylem develops during xylogenesis [37]. Xylogenesis in hypocotyls is comparable to xylogenesis in roots. Development of secondary xylem is divided here into two phases: the early phase, xylem is building from vessels and numerous parenchyma cells, and in the second, later phase, also called xylem expansion, enlarged amount of xylem elements

gymnosperms [36]; they are found also in some angiosperms, i.e., in *Populus* sp.

**2.3.** *Arabidopsis* **as a nonwoody plant example for vascular tissue formation**

grain patterning of wood in many trees [33].

116 Plant Engineering

mechanical properties of the wood [34].

detailed characterization of this process.

(PCD) process [12].

In contrast, in the *Arabidopsis* inflorescence stems stimulated mechanically by an artificial weight, the transition from primary to secondary tissue architecture leads to the development of all vasculature features mimicking secondary vascular tissues in woody plants. According to the new approach, immature inflorescence stems (9–10 cm tall) were firstly decapitated with the sharp razor blade (shoot apex and flowers were removed). Next, the artificial weight (2.5 g) was applied to the decapitated apical parts of the stems. Stems were additionally supported by a wood stick to avoid their bending. Importantly, the axillary buds grown above the leave rosettes were not removed, thus remaining the natural source of endogenous auxin. This experimental approach has been extensively described [7, 8]. It was speculated that the weight carried by the stem serves as a mechanically stimulated signal for wood formation [7, 8, 42, 51]. According to Ko and coauthors [42], mechanical stimulation of immature inflorescence stems of *Arabidopsis* increases polar auxin transport and promotes the secondary growth. It allows designing *Arabidopsis* as a full "tree-like" system. Moreover, the secondary vascular tissues develop in a very short time, namely, in 6 days [7], which is much faster than in hypocotyls [37, 38] or mature inflorescence stems of *Arabidopsis* [44, 45].

In the created *Arabidopsis* "tree-mimicking" model, the development of variety of vascular cambium phenotypes is the most spectacular. According to the obtained results, both types of cambial cells develop: (1) ray cambial cells, very short and almost round cells arranged in single-row rays mimicking transverse conducting system in woody plants, and (2) fusiform cambial cells, long, tapered-end cells, characterized by the intrusive growth and periclinal divisions, play here an important role in secondary tissue element differentiation. The phenomenon of intrusive growth of fusiform cambial cells is described for the first time in the mechanically stimulated *Arabidopsis* stems (**Figure 1A–D**), not found in the previously analyzed models. Neighboring cells start their growth in the opposite directions, but the growth is restricted to the tips of the cells, which slide along the radial cell walls and provide the elongation of the fusiform cambial cells (**Figure 1A, C**, and **D**).

New approach, based on mechanical stimulation of the immature inflorescence stems of *Arabidopsis* [7, 8, 42, 51], is expected to elucidate the phenomenon of vascular tissue formation and regeneration at cellular and molecular level—processes commonly studied in woody plants, but not fully explained yet, because of some experimental and environmental difficulties in these plants [52]. Thus, *Arabidopsis* comes out as a good model system for vascular tissue patterning.

**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).
