**2.2 Cytokinin**

*Plant Science - Structure, Anatomy and Physiology in Plants Cultured in Vivo and in Vitro*

tributes to their robustness in adverse conditions.

death of root cap cells [8, 9].

transport [15].

**2.1 Auxin**

mones, auxin functions as a central mediator.

**2. Roles of phytohormones on root formation**

wound-induced LR and AR developments [15, 24, 25].

exists throughout the plants' lifetime; it is a distinctive feature of plants and con-

In *Arabidopsis*, LRs initiate from pairs of pericycle cells that possess developmental potential as plant stem cells. These pericycle cells are selected and directed to become LR founder cells and form LRs by both intrinsic and environmental signals [2–5]. The primary LR is initiated from the basal meristem of the PR, where root cap-derived auxin influences the amplitude of oscillatory gene expression in the basal meristem and the elongation zone of the root, which leads to the prepatterning of LR initiation sites [6, 7]. The pre-patterning process is marked by the expression of a series of genes, like *GATA23*, *MEMBRANE-ASSOCIATED KINASE REGULATOR 4* (*MAKR4*), and *IAA19* [7]. In the basal meristem and elongation zone, *DR5::Luciferase* expression was observed to rhythmically pulse with a period of ~6 h, which matched with the period of LR pre-branch site production [6]. It is recently reported that the source of auxin is provided by the cyclic programmed cell

It is noteworthy that not all of the pre-branch sites emerge to be LRs [6]. The dormant pre-branch sites may present a selective mechanism for LR formation under certain growth conditions, such as water availability, nutrient levels, physical obstacles, or damage [5, 10–13]. It is interesting that many of the external signals converge on phytohormones to regulate root development. Among these phytohor-

Mechanical forces are important regulators for plant morphogenesis. LRs always emerge from the convex side of PR bending, resulting in a left-right alternation of LRs. Bending caused by gravitropic curvature led to the initiation of LRs, where a subcellular relocalization of PIN1 was observed [11]. Release the pericycle cells from the restraints of adjacent endodermis by targeted single cell ablation of endodermal cells triggered the pericycle to reenter the cell cycle and induced auxindependent LR initiation [14]. Excision of the *Arabidopsis* PR leads to the promotion of LR formation, which is mediated by activated auxin biosynthesis and auxin

The phytohormone auxin which plays fundamental roles in many aspects of plant growth and development is also a well-documented key regulator of LR development [16, 17]. The natural auxin, indole-3-acetic acid (IAA), is mainly synthesized in a two-step pathway from tryptophan. First, tryptophan is converted to indole-3-pyruvate (IPA) by the TAA1/SAV3 family of aminotransferases; IPA is then converted to IAA by the YUCCA (YUC) family of flavin monooxygenases [18–23]. Auxin biosynthesis has been shown to play an essential role on both programed and

Polar auxin transport (PAT), mediated by auxin influx (AUX1 and LAXs) and efflux carriers (PINs and MDR/PGPs) [26–29], generates auxin gradients and maintains an auxin maximum to regulate LR formation and positioning [17, 30–33]. Auxin signaling is known to be an integrator of endogenous and exogenous signals for root branching [17, 30, 34, 35]. It begins with the degradation of a class of AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) through TRANSPORT INHIBITOR RESPONSE 1 (TIR1) auxin receptor [36, 37], resulting in the activation of the AUXIN RESPONSE FACTOR (ARF) [38, 39]. ARF7 and ARF19 transcription factors further induce the expression of downstream target genes like *LATERAL* 

**22**

Cytokinin is also a main player in root development. In higher plants, isopentenyladenine (iP), trans-zeatin (tZ), and dihydrozeatin (dZ) are the predominant cytokinins [44]. Cytokinin level and patterning in plant are controlled by a fine equilibrium between cytokinin synthesis and catabolism [44, 45]. Cytokinin biosynthesis is dependent on the activity of *ATP/ADP-isopentenyltransferase* (*IPT*) and *LONELY GUY* (*LOG*) gene family [46–48], and the metabolism is mainly through the *CYTOKININ OXIDASE/DEHYDROGENASE* (*CKX*) genes [44, 45]. Cytokinin can also be inactivated through conjugation to glucose [49]. The spatial and temporal distribution of cytokinin is in part due to the specific expression of cytokinin synthesis and catabolism genes [45, 47, 48, 50–52].

In *Arabidopsis*, cytokinin signaling starts with the perception by the transmembrane cytokinin receptors ARABIDOPSIS HISTIDINE KINASE (AHK), AHK2, AHK3, and AHK4/WOL1/CRE1 [53–55], which target the histidine phosphotransfer protein AHPs to activate the type-A and type-B ARABIDOPSIS RESPONSE REGULATORS (ARRs) that negatively and positively regulate cytokinin signaling, respectively [55–61]. Two type-A ARRs, ARR7, and ARR15 were induced by both cytokine and auxin and are essential for embryonic root development [62].

Although some evidences showed that cytokinins act as both local and longdistance signals [51, 63–65], and some transporter proteins have been shown to be involved in cytokine transport [66–70], the molecular mechanisms of cytokinin transport are still not well characterized.

Postembryonic root development is regulated by the root apical meristem (RAM), where cytokinin is known to act antagonistically with auxin to control the balance of cell division in the division zone and cell differentiation in the transition zone, which is essential for the maintenance of the RAM and affects the growth and patterning of the root [64, 71]. Application of cytokinin reduces the number of meristem cells and the size of RAM and promotes cell differentiation in the transition zone; cytokinin biosynthesis and signaling mutants as well as *CKX* overexpression mutants have a larger RAM with more meristem cells [45, 64, 72]. Conversely, auxin treatment increases meristem size and promotes cell division in the proximal meristem, and auxin transporter *PIN* mutants display a smaller RAM [64, 73]. The cross-talk of cytokine and auxin in regulating RAM activity was shown to converge on the auxin-inducible *AUX/IAA* family gene *SHORT HYPOCOTYL 2/ INDOLE-3-ACETIC ACID 3* (*SHY2/IAA3*) in the transition zone, where cytokinin activates *SHY2* via the *AHK3/ARR1* two-component signaling pathway to suppress *PIN3* and *PIN7* expression and promote cell differentiation, while auxin suppressed SHY2 protein, leading to the activation of PINs and promotion of cell division [71]. Furthermore, Moubayidin et al. [74] revealed that in transition zone, SCR, a member of the GRAS family of transcription factors, directly represses the expression of *ARR1*, which controls auxin production via the *ASB1* gene and sustains stem cell activity, to simultaneously control stem cell division and differentiation and ensure coherent root growth. Cytokinin affects the expression of multiple *PINs* differentially in a tissue-specific manner to regulate auxin distribution [75, 76]. Auxin-cytokinin interactions lead the generation of distinct hormonal response zones, thus controlling the development of root vascular tissue.

On contrary to auxin, which is a positive regulator of LR development, cytokinin acts as a negative regulator of LR formation. Cytokinin suppresses LR initiation

through downregulating *PIN* expression and preventing the establishment of auxin gradient in LR founder cells [77]. Mutants with reduced cytokinin level or deficient cytokinin signaling increased the number of LRs [45, 58, 60, 78], while cytokinin treatment suppresses LR initiation and development [77, 79, 80]. Li et al. [80] reported that cytokinins inhibit LR initiation by blocking the cell cycle of pericycle founder cell at G2 to M transition phase while promoting LR elongation by stimulation of the G1 to S transition.

Through mutant analysis Chang et al. [81] showed that cytokinin biosynthesis genes *IPT3* and *IPT5* and all three cytokinin receptor genes *AHK2*, *AHK3*, and *CRE1/AHK4* act redundantly during LR initiation, and early stages of lateral root primordia (LRP) formation are particularly cytokinin sensitive. They suggest that cytokinin may serve as a positional signal for new LRP formation. In rice, ERF3 interacts with WOX11 to promote crown root initiation and elongation by regulating the cytokinin-responsive gene *RR2* [82]. Cytokinin has also been shown to modulate LR formation by mediating environmental cues. Jeon et al. [83] showed that CYTOKININ RESPONSE FACTOR 2 (CRF2) and CRF3 encoding APETALA2 transcription factors regulate *Arabidopsis* LR initiation under cold stress.

#### **2.3 Other phytohormones**

Other phytohormones, like abscisic acid (ABA), gibberellic acid (GA), brassinosteroid (BR), jasmonic acid (JA), ethylene, and strigolactone (SL), also participate in root growth and development.

Signora et al. [84] showed that ABA plays an important role in mediating the effects of nitrate on LR formation in *Arabidopsis*. Brady et al. [85] reported that *ABSCISIC ACID INSENSITIVE 3* (*ABI3*) is involved in auxin signaling and LR development. De Smet et al. [86] reported that ABA application leads to the inhibition of LR development immediately after the emergence of the LRP from the parent root and prior to the activation of the LR meristem in an auxin-independent manner. Shkolnik-Inbar and Bar-Zvi [87] showed that *ABI4*, which encodes an ABA-regulated AP2 domain transcription factor, mediates ABA and cytokinin inhibition of LR formation through the reduction of PAT. Ding et al. [88] reported that ABA signaling and auxin homeostasis regulate WRKY46 to modulate the development of LR in osmotic/salt stress condition.

Hansen [89] reported on the GA-mediated light dependent promotion and inhibition of AR formation. Through mutant analysis, Yaxley et al. [90] showed that GA is important for normal root elongation in pea. Fu and Harberd [91] showed that auxin regulates root growth through GA-mediated DELLA protein destabilization. Steffens et al. [92] showed that GA is ineffective on its own but acts synergistically with ethylene to promote the number of penetrating roots and the growth rate of emerged roots in deepwater rice.

BR is a positive regulator of root development [93]. Bao et al. [94] showed that BRs interact with auxin to promote LR development.

JA, a crucial plant defense hormone, also participated in the regulation of root development. Raya-González et al. [95] observed that low concentrations of JA inhibited PR growth through an auxin-independent manner and promoted LR formation auxin-dependently, and JA receptor COI1 is involved in JA-induced LR formation and LR positioning. Cross-talk between JA and auxin has been frequently reported. JA has been reported to be implicated in YUC9-mediated auxin biosynthesis in wounded leaves in *Arabidopsis* [96]. Cai et al. [97] also reported a crosstalk between JA and auxin biosynthesis during LR formation mediated by ERF109. Gutierrez et al. [98] showed that auxin controls *Arabidopsis* AR initiation through the regulation of JA homeostasis.

**25**

*Phytohormone-Mediated Homeostasis of Root System Architecture*

Ethylene is also a well-known phytohormone that participates in the plant defense signaling pathways. Strader et al. [99] reported that ethylene interact with auxin to control root cell expansion. Ivanchenko et al. [100] observed application of low level of ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) promotes LRP initiation, while higher doses of ACC strongly inhibits LRP initiation but promotes LRP emergence; this regulation of LR initiation and emergence by ethylene is through interactions with auxin. Lewis et al. [101] reported that ethylene suppresses LR formation through promotion of PIN3 and PIN7-mediated auxin

Jiang et al. [102] showed that SL analog GR24 negatively influenced LR priming and emergence, which is dependent on the intimate connection with auxins and

The root system of higher plants is modified by intrinsic developmental signals and diverse environmental cues. Both the internal and the external signals converged on phytohormones to regulate the formation of a highly plastic and adaptive RSA, which sustains the growth of plants even in adverse conditions. Several lines of evidences suggest that cross-talks among different phytohormones are essential for the regulation of root development, and auxin plays a central role in these processes. Although auxin and cytokinin as the key regulators of root development have been extensively studied, the roles of other phytohormones still need to be further characterized to give us a full view of

*DOI: http://dx.doi.org/10.5772/intechopen.82866*

efflux to prevent local auxin accumulation.

**3. Conclusions**

plant root development.

**Conflict of interest**

**Acronyms and abbreviations**

PR primary root LR lateral root AR adventitious root IAA indole-3-acetic acid PAT polar auxin transport iP isopentenyladenine tZ trans-zeatin dZ dihydrozeatin RAM root apical meristem LRPs lateral root primordia

ABA abscisic acid GA gibberellic acid BR brassinosteroid JA jasmonic acid SL strigolactone

RSA root system architecture

cytokinins, with the PAT capacity as a central player.

The authors declare no conflict of interest.

ACC 1-aminocyclopropane-1-carboxylic acid

*Phytohormone-Mediated Homeostasis of Root System Architecture DOI: http://dx.doi.org/10.5772/intechopen.82866*

Ethylene is also a well-known phytohormone that participates in the plant defense signaling pathways. Strader et al. [99] reported that ethylene interact with auxin to control root cell expansion. Ivanchenko et al. [100] observed application of low level of ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) promotes LRP initiation, while higher doses of ACC strongly inhibits LRP initiation but promotes LRP emergence; this regulation of LR initiation and emergence by ethylene is through interactions with auxin. Lewis et al. [101] reported that ethylene suppresses LR formation through promotion of PIN3 and PIN7-mediated auxin efflux to prevent local auxin accumulation.

Jiang et al. [102] showed that SL analog GR24 negatively influenced LR priming and emergence, which is dependent on the intimate connection with auxins and cytokinins, with the PAT capacity as a central player.
