The molecular path to in vitro shoot regeneration.

JBA-06771; No of Pages 15 Biotechnology Advances xxx (2013) xxx–xxx

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Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

The molecular path to in vitro shoot regeneration Hans Motte a,b,1,2, Danny Vereecke a,1, Danny Geelen b,⁎, Stefaan Werbrouck a a b

Department of Applied Biosciences, Faculty of Bioscience Engineering, Ghent University, V. Vaerwyckweg 1, BE-9000 Ghent, Belgium Department of Plant Production, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, BE-9000 Ghent, Belgium

a r t i c l e Available online xxxx Keywords: Organogenesis Auxin Cytokinin Tissue culture

i n f o

a b s t r a c t Plant regeneration through de novo shoot organogenesis in tissue culture is a critical step in most plant transformation and micropropagation procedures. Establishing an efficient regeneration protocol is an empirical process and requires optimization of multiple factors that influence the regeneration capacity. Here, we review the molecular process of shoot induction in a two-step regeneration protocol and focus on the role of auxins and cytokinins. First, during incubation on an auxin-rich callus induction medium (CIM), organogenic callus is produced that exhibits characteristics of a root meristem. Subsequent incubation on a cytokinin-rich shoot induction medium (SIM) induces root to shoot conversion. Through a detailed analysis of the different aspects of shoot regeneration, we try to reveal hinge points and novel candidate genes that may be targeted to increase shoot regeneration capacity in order to improve transformation protocols. © 2013 Elsevier Inc. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The two-step regeneration protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Auxin initiates the formation of organogenic callus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Local auxin accumulation specifies founder cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Auxin signaling in regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Callus and primordium initiation are accompanied by changes in expression of genes involved in lateral root formation 3.4. Acquisition of shoot competence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Cytokinin-mediated cell fate respecification and establishment of shoot meristem organization . . . . . . . . . . . . . . 4.1. Uptake of cytokinins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Cytokinin biosynthesis and metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Cytokinin signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Cytokinin-induced WUSCHEL expression marks the onset of shoot meristem formation . . . . . . . . . . . . . . 4.5. Auxin-cytokinin crosstalk and auxin transport are important factors controlling shoot meristem organization . . . . 4.6. CUC-STM interplay during shoot initiation and development . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7. WUS, STM and shoot determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ⁎ Corresponding author. Tel.: +32 9 264 60 76; fax: +32 9 264 62 25. E-mail addresses: [email protected] (H. Motte), [email protected] (D. Vereecke), [email protected] (D. Geelen), [email protected] (S. Werbrouck). 1 Equal contribution. 2 Present address: Department of Plant Systems Biology, VIB, BE-9052 Ghent, Belgium; and Department of Biotechnology and Bioinformatics, Ghent University, BE-9052 Ghent, Belgium.

Plant transformation is achieved by the transfer of DNA to the genome of a plant cell, the subsequent development of a shoot from the transgenic cell, and, finally, the formation of a root system to render the genetically modified plant complete. Agrobacterium-based methods or particle bombardment are largely the preferred DNA transfer techniques (for reviews, see Păcurar et al., 2011; Rao et al., 2009; Vyacheslavova et al., 2012). Although transgenic plants can be obtained

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Please cite this article as: Motte H, et al, The molecular path to in vitro shoot regeneration, Biotechnol Adv (2013), http://dx.doi.org/10.1016/ j.biotechadv.2013.12.002

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H. Motte et al. / Biotechnology Advances xxx (2013) xxx–xxx

directly via seeds, for instance after transformation of gametes (Bechtold et al., 1993; Clough and Bent, 1998; Luo and Wu, 1989), this technique is not widely applicable (Rao et al., 2009). Generally, plant regeneration from transformed cells is a required step in transformation protocols and involves respecification of cell fate and coordinated cell divisions (Zhao et al., 2008). Plant regeneration can be attained through somatic embryogenesis or de novo shoot organogenesis via in vitro tissue culture techniques (Sugimoto et al., 2011). While the molecular process of somatic embryogenesis is still poorly understood (for reviews, see Namasivayam, 2007; Verdeil et al., 2007; Yang and Zhang, 2010), the molecular path to shoot regeneration has steadily been revealed (Duclercq et al., 2011b; Meng et al., 2010). The remarkable ability of plant cells to regenerate can be traced back to the totipotency theory first hypothesized by Haberlandt (1902): “Theoretically all plant cells are able to give rise to a complete plant”. Whereas Haberlandt succeeded only in the survival of in vitro grown tissue, Hannig (1904) obtained in vitro plant cell division, and Simon (1908) was the first to regenerate buds on callus tissue. However, it took half a century to control organogenesis: Skoog and Miller (1957) induced organ differentiation by changing the relative concentrations of auxin and cytokinin in the medium. Since then, based on their experiments, shoot regeneration has been established for many plants, with applications for instance in micropropagation, mutagenesis, and polyploidization. In the 1980's, major achievements in plant transgenic research led to an important breakthrough in plant science (reviewed by Van Montagu, 2011), resulting in an enormous expansion of tools for basic research and in numerous agricultural applications, for example in improvement of traits, such as biotic and abiotic stress tolerance, yield, nutritional value, etc. (Ahmad et al., 2012; Vyacheslavova et al., 2012). Moreover, because plant regeneration was an essential step to obtain transgenic plants, a new and very important application for shoot organogenesis had arisen. The experiments of Skoog and Miller gave many insights in shoot regeneration, but their relatively simple findings could not straightforwardly be adopted for other plants. Indeed, a series of events occurs before shoot regeneration takes place (Cary et al., 2002; Che et al., 2007; Christianson and Warnick, 1983) and, depending on the plant species and explants used, different treatments have to be applied. Moreover, although for some plants root formation occurs spontaneously, for others it is difficult or even impossible to achieve (Geiss et al., 2009; Oinam et al., 2011). Thus, the development of efficient shoot and root regeneration protocols for many economically important plants remains cumbersome and mainly a matter of trial and error, and therefore, little or no transgenics have been obtained for these plants. Although somatic embryogenesis and root production are important aspects of plant regeneration, in this review we will focus on shoot regeneration, because in the last two decades significant progress has been made in the understanding of the developmental and molecular mechanisms underlying this process. These advances are the combined merit of the rise of the model plant Arabidopsis thaliana, the fast development of new molecular techniques (Duclercq et al., 2011b; Meng et al., 2010), and intensive research on general plant development in the field of auxin and cytokinin. Here, we will give a chronological overview of the processes that occur during the establishment of de novo shoot formation in Arabidopsis following a two-step regeneration protocol starting from root explants and combine this with information on organ formation in intact plants. The successive events and the genes involved are summarized in Fig. 1 and the impact of misexpressing these genes on shoot regeneration is given in Table 1. With this, we will try to pinpoint possible hinge points that may be at the basis of regeneration recalcitrance and highlight particular findings that might help to improve regeneration and transformation protocols. 2. The two-step regeneration protocol In vitro organogenesis is a multistep process consisting of founder cell specification, callus/primordium formation, acquisition of organogenesis

competence, assignment of organ identity to the developing primordia, and, finally, development of the organ. The latter aspect will not be dealt with in this review, because it does not differ from organ outgrowth in intact plants. In efficient shoot regeneration protocols, including those for Arabidopsis, this sequence of events is generally accomplished by two subsequent incubation steps: first treatment of the explant on an auxin-rich callus induction medium (CIM), followed by incubation on a cytokinin-rich shoot induction medium (SIM). During this two-step protocol, founder cell specification, the development of primordia and the acquisition of organogenesis competence are mediated by auxin, whereas the assignment of the shoot identity to the developing primordia is controlled by cytokinin (Fig. 1). From this point onward, we will focus on the two-step shoot regeneration protocol as described by Valvekens et al. (1988), applied on root explants from Arabidopsis. During incubation on CIM, founder cell specification, or the assignment of a particular subset of cells to give rise to an organ (Chandler, 2011), occurs in the pericycle cells (Atta et al., 2008; Che et al., 2007). Historic anatomical studies on Convolvulus arvensis roots demonstrated that the initial cell divisions of the pericycle cells, their redifferentiation into founder cells, and the subsequent morphogenic events leading to primordium formation preceding shoot development, strongly resembled those occurring during lateral root formation (Beijerinck, 1887; Bonnett and Torrey, 1966). These findings were recently corroborated in Arabidopsis by using molecular markers and led to the rejection of the generally accepted assumption that callus is composed of dedifferentiated cells. The new consensus is that root-derived callus, as well as leaf- and hypocotyl-derived callus, resembles a root primordium (Atta et al., 2008; Sugimoto et al., 2010). These calli or very young primordia, in se defined as undifferentiated organ precursors in their earliest recognizable stage of development, acquire the competence to form organs, i.e. the ability to respond to signals that direct the formation of a particular organ (Cary et al., 2002; Che et al., 2007; Sugiyama, 1999). At this stage, they can give rise to roots as well as shoots, and should thus be considered as organ primordia. The tissue obtained after CIM incubation is termed organogenic callus. Depending on the subsequent culture conditions, the organ primordia of the organogenic callus will irreversibly develop either into a root or a shoot (Atta et al., 2008; Christianson and Warnick, 1983). When the explants are kept on auxin-rich medium, the developing primordia will obtain a determined root identity and lose the ability to form shoots. Conversely, when they are transferred to cytokinin-rich SIM, the primordia will get a determined shoot identity (Cary et al., 2002; Christianson and Warnick, 1983; Gordon et al., 2009). In the subsequent developmental steps that occur on SIM, auxin–cytokinin crosstalk is particularly important during patterning of the shoot primordium and the shoot meristem (Besnard et al., 2011; Cheng et al., 2013; Gordon et al., 2007; Zhao et al., 2010). Because in a wide range of plants, comparable anatomical and developmental stages can be discerned during shoot regeneration as in Arabidopsis (eg. Attfield and Evans, 1991; Beijerinck, 1887; Bonnett and Torrey, 1966; Christianson and Warnick, 1983; Flinn et al., 1988; Peterson, 1970; Rocha et al., 2012; Spencer-Barreto and Duhoux, 1994; Vila et al., 2005; Vinocur et al., 2000) and since the auxin and cytokinin machineries are highly conserved (De Smet et al., 2011; Finet and Jaillais, 2012; Spíchal, 2012), the principle of the two-step regeneration protocol is adaptable to many plants. Therefore, it is a good model system to obtain general insights on shoot regeneration. 3. Auxin initiates the formation of organogenic callus When root explants are placed on CIM, pericycle cells start to divide giving rise to founder cells and ultimately to organ primordia with the competence to form shoots. Because shoot and lateral root formation in intact roots share their initial developmental stages (Atta et al., 2008; Sugimoto et al., 2010), insights from lateral root research could be informative to comprehend the sequence of events that occurs on



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Fig. 1. Overview of the molecular events and the genes involved in the two-step shoot regeneration process from Arabidopsis root explants. Full names of the genes are given in the text. References are given in Table 1 and in the text. Incubation periods of CIM and SIM are based on the Col-0 accession.

CIM. Indeed, mutants such as aux1, iaa3/shy2-2, iaa14/slr, and alf4 that lack the capacity for efficient lateral root induction, (Péret et al., 2009, and refs therein), also show a decreased regeneration capacity (Table 1). In the following subsections, we will first describe the initial steps of lateral root formation in order to relate them to events occurring on CIM. 3.1. Local auxin accumulation specifies founder cells In intact roots, the earliest detectable events in founder cell specification is the establishment of local auxin maxima and the concomitant local activation of auxin responses in specific pericycle cells (Dubrovsky et al., 2008), controlled by auxin influx and efflux carriers (Löfke et al., 2013; Ruiz Rosquete et al., 2012). This is necessary and sufficient to respecify these cells into founder cells of root primordia, which will result in lateral root formation (Benková et al., 2003). Several lines of evidence show that pericycle cell division driven by local auxin maxima is essential for shoot regeneration as well. For instance, loss of the pericycle cells by ablation prevents shoot regeneration (Che et al., 2007). Moreover, the particular characteristics of the synthetic auxins that are commonly used in CIM, such as 2,4-

dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA), make them very efficient for generating multiple auxin maxima throughout the explants. 2,4-D is not transported out of the cells by the PIN-FORMED (PIN) efflux carriers (Delbarre et al., 1996) and is only very poorly metabolized (Hošek et al., 2012), which allows accumulation in the cells. NAA on the other hand, can enter the cells independent from the AUX/LAX auxin influx carriers (Delbarre et al., 1996) and is likewise metabolized more slowly than natural auxins (Beyer and Morgan, 1970), rendering it more persistent in the explant. Although the auxin influx carriers AUXIN-RESISTANT1 (AUX1), LIKE AUX1-1 (LAX1), LAX2 and LAX3, contribute to the local auxin accumulation during the first stage of shoot regeneration, the malfunctioning of these genes can easily be circumvented. For instance, root explants of the aux1 mutant are not capable of forming callus under standard culture conditions, but by increasing the auxin concentration in the CIM, callus is formed and regeneration takes place (Kakani et al., 2009). Probably, redundancy between the different influx carriers assures a sufficient auxin supply and, consequently, local auxin accumulation (Bainbridge et al., 2008). The PIN auxin efflux carriers on the other hand, important in generating auxin gradients during organ formation (Benková et al., 2003), appear to negatively affect regeneration


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Table 1 Genes involved in regeneration-related processes, of which overexpression (OE), induced expression (IE), and/or loss of function (LOF) alters the shoot regeneration capacity. Genea

Process

Phenotype

Reference

AUX1 YUCs TIR1 SLR/IAA14 LBD16, LBD17, LBD18, LBD29

Auxin influx Auxin biosynthesis Auxin perception Auxin signaling Auxin signaling

(Kakani et al., 2009) (Cheng et al., 2013; Zhao et al., 2001) (Qiao et al., 2012b) (Atta et al., 2008) (Fan et al., 2012)

IAA3/SHY2

Auxin signaling

ALF4 IPTs

Lateral root cell division Cytokinin biosynthesis

CYP735A2 CKXs GLU AHK2, AHK3 AHK4 CKI1 Type B ARRs

trans-zeatin biosynthesis Cytokinin degradation Cytokinin activation Cytokinin perception Cytokinin perception Cytokinin signaling Cytokinin signaling

CRFs Type C ARRs Type A ARRs PIN1

Cytokinin signaling Cytokinin signaling Cytokinin response Shoot-related auxin transport

LOF requires increased auxin level for callus formation OE eliminates CIM requirement; LOF decreases regeneration capacity LOF decreases and OE increases regeneration capacity OE decreases regeneration capacity OE induces spontaneous callus formation with efficient regeneration capacity; LOF inhibits callus formation on CIM OE suppresses the formation of shoot meristems; LOF induces CUC expression LOF blocks callus formation IE induces spontaneous shoot formation; LOF decreases regeneration capacity Presumable IE induces STM-marked meristems on leaves OE decreases regeneration capacity OE increases regeneration capacity LOF decreases regeneration capacity LOF blocks shoot formation OE induces cytokinin-independent shoot formation OE induces cytokinin-independent shoot formation; LOF decreases regeneration capacity LOF decreases regeneration capacity OE blocks shoot formation LOF increases and OE decreases regeneration capacity LOF decreases regeneration capacity

PID ESRs

Shoot-related auxin transport Shoot-related auxin transport

ARF3 ARF10,16,17 CUCs LSHs

Cytokinin controlling auxin signaling Shoot-related auxin signaling Shoot development Shoot development

STM

Shoot development

WUS

Shoot development

ETR1, EINs CTRs, HLS1 ETO1 CDKB2s WIND1 PHYs, CRYs, HY5 ATHB15 RAP2.6L FLA1 GLB1, GLB2

Ethylene signaling Ethylene response Ethylene biosynthesis inhibition Cell division Wound response Light sensing Unknown Unknown Unknown Unknown

a

LOF decreases regeneration capacity LOF decreases and OE increases regeneration capacity and induces CUC expression LOF decreases regeneration capacity LOF decreases and OE increases regeneration capacity LOF decreases and OE increases regeneration capacity OE induces WUS-expressing meristem-like tissues, WUS- and STM-expressing shoot-like primordia and shoots on flowers IE induces multiple ectopic shoot meristems, LOF blocks shoot formation LOF blocks shoot formation or decreases shoot regeneration and OE increases regeneration capacity LOF decreases regeneration capacity LOF increases regeneration capacity LOF increases regeneration capacity OE blocks shoot formation OE induces CIM-independent shoot formation LOF decreases regeneration capacity Protein modification induces cytokinin-independent shoot formation LOF decreases regeneration capacity LOF decreases regeneration capacity LOF decreases and OE increases regeneration capacity

(Koyama et al., 2010) (Sugimoto et al., 2010) (Cheng et al., 2013; Kunkel et al., 1999) (Uchida et al., 2011) (Yang et al., 2003) (Klemš et al., 2011) (Nishimura et al., 2004) (Nishimura et al., 2004) (Hwang and Sheen, 2001) (Hwang and Sheen, 2001; Ishida et al., 2008) (Rashotte et al., 2006) (Kiba et al., 2004) (Buechel et al., 2010) (Cheng et al., 2013; Gordon et al., 2007) (Matsuo and Banno, 2012) (Banno et al., 2001; Ikeda et al., 2006; Matsuo et al., 2011) (Cheng et al., 2013) (Qiao et al., 2012a) (Daimon et al., 2003) (Takeda et al., 2011) (Brand et al., 2002; Daimon et al., 2003) (Chatfield et al., 2013; Gallois et al., 2004; Gordon et al., 2007) (Chatfield and Raizada, 2008) (Chatfield and Raizada, 2008) (Chatfield and Raizada, 2008) (Andersen et al., 2008) (Iwase et al., 2011) (Nameth et al., 2013) (Duclercq et al., 2011a) (Che et al., 2006) (Johnson et al., 2011) (Wang et al., 2011b)

Full names are given in the text.

at this early stage, since the inhibition of polar auxin transport was shown to stimulate organogenic callus formation (Pernisová et al., 2009). Later in the regeneration process, PINs are required for shoot morphogenesis (Cheng et al., 2013; Gordon et al., 2007), which will be discussed further in Section 4.5. Finally, the endogenous auxin level of the explant has a strong influence on the regeneration capacity. Indeed, Arabidopsis lines or mutants with a high expression of the auxin biosynthetic YUCCA (YUC) genes have a high regeneration capacity even in protocols without CIM incubation (Zhao et al., 2001, 2013). In contrast, potato plants with a naturally high endogenous auxin concentration produce only callus in conventional regeneration protocols and require an anti-auxin for regeneration (Pal et al., 2012). Thus, screening novel auxins, auxin-like compounds, and/or auxin transport modulators for the efficient establishment of multiple auxin maxima might be a successful approach for improving regeneration efficiency. 3.2. Auxin signaling in regeneration Auxin signaling starts with the perception of the accumulating auxin by the SCFTIR1/AFB1–5 complexes, including the F-box auxin co-receptor

proteins TRANSPORT INHIBITOR RESISTANCE1 (TIR1) and AUXIN F-BOX BINDING1-5 (AFB1–5), which triggers the degradation of Aux/IAA transcriptional repressors (Calderón Villalobos et al., 2012; Dharmasiri et al., 2005a,b; Greenham et al., 2011; Kepinski and Leyser, 2005; Tan et al., 2007). Subsequently, AUXIN RESPONSE FACTORS (ARFs), released from these inhibitors, mediate auxin-dependent gene expression (Ulmasov et al., 1997, 1999). Different Aux/IAA-ARF modules are crucial for several successive events during lateral root initiation. (i) Auxin accumulation in pericycle cells leads to the degradation of the Aux/IAA28 transcriptional repressor which controls the founder cell-specifying GATA23 transcription factor (De Rybel et al., 2010). (ii) Division of the founder cells for organogenesis initiation is dependent on the auxin-activated degradation of SOLITARY ROOT (SLR/IAA14) (Fukaki et al., 2002) and the subsequent activation of ARF7 and ARF19 (Okushima et al., 2005). These ARFs are negatively regulated by PICKLE/SUPPRESSOR OF SLR2 (PKL/SSL2) (Fukaki et al., 2006) and regulate the transcriptional activation of LATERAL ORGAN BOUNDARIES-DOMAIN29/ASYMMETRIC LEAVES2-LIKE16 (LBD29/ ASL16), LBD16/ASL18 and LBD18/ASL20, that function in the initiation and emergence of lateral roots (Lee et al., 2009; Okushima et al., 2007). LBD16/ASL18 is specifically expressed in founder cells resulting



in nuclear migration and subsequent asymmetric division of each founder cell which preludes organogenesis (Goh et al., 2012a). (iii) After the first cell division and activation of the SLR/IAA14-ARF7/19 auxin response module, the BODENLOS (BDL)/IAA12-MONOPTEROS (MP)/ ARF5—mediated auxin response is required for root organogenesis (De Smet et al., 2010). At the same time, the receptor-like kinase ARABIDOPSIS CRINKLY4 (ACR4) is transcribed specifically in the small daughter cells of the asymmetrically divided founder cell and suppresses proliferative cell divisions in nearby pericycle cells (De Smet et al., 2008). The regulation of ACR4 expression requires SLR/IAA14 (De Smet et al., 2008; Vanneste et al., 2005). In addition, SHORT HYPOCOTYL2/SUPPRESSOR OF HY2 (SHY2)/IAA3 is important for the emergence of the root primordium (Swarup et al., 2008). Clearly, auxin perception and the activation of several auxin signaling modules are consecutively and sometimes simultaneously required during lateral root initiation. If these stages are shared with organogenic callus or organ primordium formation, one should expect that defects in auxin signaling would cause regeneration recalcitrance. Indeed, mutations in several of the discussed genes interfere with primordium initiation or shoot organogenesis and overexpression of the downstream genes is in these cases sufficient to restore the wild-type phenotype (Table 1). For example, loss of function mutations in TIR1 decrease, while TIR1 overexpression increases the shoot regeneration capacity (Qiao et al., 2012b). Gain of function mutations of SLR/IAA14 (Fukaki et al., 2002; Vanneste et al., 2005) and arf7 arf19 double mutations (Okushima et al., 2005, 2007; Wilmoth et al., 2005) prevent the initiation of lateral root primordia, but also result in a reduced shoot regeneration (Atta et al., 2008). Moreover, ectopic expression or suppression of the LBD genes enhances or inhibits callus formation, respectively, leading to an altered shoot regeneration capacity (Fan et al., 2012) and aberrant lateral root formation4 (alf4) mutants incapable of lateral root initiation are unable to form callus (Sugimoto et al., 2010). Altogether, the altered regeneration phenotypes of these mutants underline the functional overlap of the early events in lateral root initiation and shoot regeneration. The exploration of lateral root mutants with defects in other auxin-related genes or other auxin-signaling modules (reviewed by Péret et al., 2009) than the ones discussed here, might reveal novel genes and processes involved in shoot regeneration. Thus, one of the challenges for regeneration research is to unravel the exact role of each part of the auxin machinery in primordium and shoot formation. 3.3. Callus and primordium initiation are accompanied by changes in expression of genes involved in lateral root formation During lateral root development, when auxin accumulates in the pericycle cells, the root quiescent center marker WUSCHEL-RELATED HOMEOBOX5 (WOX5) is expressed in the founder cells (Ditengou et al., 2008). The subsequent development of the lateral root primordia consists of a series of anticlinal cell divisions, followed by periclinal divisions (Casimiro et al., 2003), which are accompanied by expression of the root stele marker SHORT-ROOT (SHR) (Lucas et al., 2011). In the primary root tip, SHR activates SCARECROW (SCR), which marks the root endodermis and quiescent center (Levesque et al., 2006) and the AP2 transcription factor PLETHORA1 (PLT1) is expressed downstream of WOX5 (Ding and Friml, 2010). Then, through the concerted action of PLT1, SHR, and SCR, the PIN genes are expressed (Xu et al., 2006). Similar polarized PIN accumulation establishes local auxin maxima at the tip of the lateral root primordia (Benková et al., 2003). During the CIM incubation phase of the shoot regeneration process, many of the genes involved in lateral root initiation are induced as well, demonstrating the strong commonalities in the development of lateral root primordia and the primordia that will ultimately give rise to shoots. For instance, WOX5 is expressed in the subepidermal layer of the callus, SHR and SCR are expressed throughout the callus (Sugimoto et al., 2010), and PLT1 and PIN1 are activated in the organ primordia (Atta

5

et al., 2008; Gordon et al., 2007). Moreover, expression of ROOT CLAVATA HOMOLOG1 (RCH1) and QUIESCENT CENTER25 (QC25), described as root apical meristem markers (Casamitjana-Martinez et al., 2003; Sabatini et al., 2003), is detected in the callus induction phase as well (Atta et al., 2008), and GLABRA2 (GL2), marking the non-hair epidermal cells in the meristematic zone of the root (Lin and Schiefelbein, 2001), show a similar striped expression pattern in callus and roots (Sugimoto et al., 2010). Interestingly, the cytokinin biosynthesis gene ISOPENTENYLTRANSFERASE5 (IPT5) and the cytokinin response gene ARABIDOPSIS RESPONSE REGULATOR5 (ARR5) exhibit a comparable local expression pattern in the lateral root primordia and in the organ primordia formed on CIM (Atta et al., 2008; D'Agostino et al., 2000; Miyawaki et al., 2004), suggesting a role for cytokinin in the very early stages of shoot regeneration. With the availability of several genome-wide transcript datasets from callus formation (Che et al., 2006; He et al., 2012; Sugimoto et al., 2010; Xu et al., 2012) and from specific root cells or tissues involved in lateral root initiation (Brady et al., 2007; De Smet et al., 2008; Himanen et al., 2004; Vanneste et al., 2005), comparative metaanalyses could shed more light on the common pathways of the early stages of shoot regeneration and lateral root formation. As such, Sugimoto et al. (2010) noticed that about one third of the genes upregulated during callus induction, were actually genes upregulated in the tip zone of the root (Brady et al., 2007). In an additional meta-analysis, we compared the dataset of Xu et al. (2012), who profiled the transcriptome of callus at different time points with short intervals during the first 4 days of CIM incubation, with two datasets specific for lateral root induction, the transcriptome of dividing pericycle cells undergoing lateral root initiation (De Smet et al., 2008) and typical lateral root initiation genes deduced from transcriptional analysis of the slr1 mutant (Vanneste et al., 2005). From the former, we only included the non-transiently up-regulated gene clusters 1, 2, 3 and 9 (for details, see De Smet et al., 2008), retaining 1109 genes. Interestingly, the majority of both sets of lateral root initiation genes overlapped with the callus-related genes (Fig. 2A): 847 of the 1109 genes upregulated in the dividing pericycle cells (De Smet et al., 2008) and 643 of the 913 genes identified as lateral root initiation genes (Vanneste et al., 2005) are also upregulated (N2-fold) during CIM incubation, supporting the involvement of root-related genes during callus formation. Next, we conducted a more time-specific comparison, as this could reveal more insights into the kinetics of the root-like gene expression program during callus induction. Therefore, the number of CIM-induced genes was plotted for each individual time point on a bar chart (Fig. 2B): we distinguished between the total number of induced genes (yellow bars) and the fraction of lateral root initiation-related genes (green and purple bars). When the total number of upregulated genes is considered over time (yellow bars), the cumulative curve (yellow dashed line) has a steep slope, illustrating that there is an enrichment of new genes at each time point and that the overall transcriptome is variable in time. In contrast, when we consider the upregulated lateral rootrelated genes (green and purple bars), the difference between the different time points is much lower, implying that with time only a limited number of additional upregulated genes contribute to the process and that most of the genes upregulated in one phase remain upregulated throughout CIM incubation. Especially from 24 h onward, these cumulative curves reach almost perfect plateaus (green and purple dashed lines), suggesting that from then on, a fixed root-like gene expression program is established. Remarkably, 24 h is also the minimal CIM incubation period required for the acquisition of competence of callus (Che et al., 2007). Thus, the genes exhibiting this root-like expression program are potentially important for callus and subsequent shoot formation. Interestingly, although several of these genes, strongly induced on CIM (N 10-fold) and also induced in both lateral root-related datasets, have proven to be essential during lateral root initiation and the initial stages of regeneration, many of these genes have not been described in this context (Table 2). Therefore,


6


A

CIM

LRI

(Xu et al., 2012)

(Vanneste et al., 2005)

LRI pericycle (De Smet et al., 2008)

B 4500

Number of upregulated genes (> 2-fold)

all 4000

dividing pericycle cell genes

3500

lateral root initiation genes

3000 2500 2000 1500 1000 500 0 12

24

48

96

Time of CIM-incubation (h) Fig. 2. Meta-analysis of a CIM-induced (Xu et al., 2012) and two lateral root initiationrelated transcriptomes (De Smet et al., 2008; Vanneste et al., 2005). (A) Venn diagram representing the overlap between the three datasets. The majority of lateral root initiation-related genes are also CIM-induced. (B) Bar chart representing the number of genes upregulated (N2-fold) after 12, 24, 48, and 96 h of CIM incubation. Yellow bars represent all upregulated genes (Xu et al., 2012), while green and purple bars only consider upregulated genes in dividing pericycle cells (De Smet et al., 2008) or lateral root initiation genes (Vanneste et al., 2005), respectively. Dashed lines represent the cumulative number of upregulated genes. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

these genes might represent novel players in shoot regeneration and they merit further study to unravel their putative role in this developmental process. 3.4. Acquisition of shoot competence The ultimate goal of incubating explants on CIM is to obtain organogenic callus with primordia that have the competence to form organs. In Arabidopsis this takes at least two days of CIM incubation (Che et al., 2007; Motte et al., 2011). Interestingly, although WUSCHEL (WUS), a critically important gene for shoot regeneration (see below), is not expressed on CIM, a 48 h CIM pre-treatment is required for WUS to be expressed during SIM incubation (Che et al., 2007). Consequently, markers for the acquisition of organogenesis competence should be expressed during CIM incubation. A possible candidate is the NAC transcription factor CUP-SHAPED COTELYDON2 (CUC2), of which the transcript locally accumulates after two days of CIM incubation at sites with the potential to form shoots (Motte et al., 2011). Similarly, the cytokinin receptor ARABIDOPSIS HISTIDINE KINASE4 (AHK4) might also

be a marker. Indeed, during CIM incubation, localized AHK4 expression identifies sites of future cytokinin-induced WUS transcription during the subsequent incubation on SIM (Gordon et al., 2009). Again, this suggests a possible role of cytokinin and cytokinin signaling early in the regeneration process. Finally, Qiao et al. (2012a) identified a number of microRNAs with a differential expression in regenerative and recalcitrant callus. These factors or their targets might be necessary for downstream shoot development on SIM. Indeed, miR165 and miR166 were shown to be important during shoot induction and development (Jung and Park, 2007; Liu et al., 2013; Zhang and Zhang, 2012). In tomato, REGENERATION1 (RG1), a gene identified by the characterization of a highly regenerative natural variant (Koornneef et al., 1993), and PROCERA (PRO) (Bassel et al., 2008), are proposed to be key genes in the acquisition of shoot competence (Lombardi-Crestana et al., 2012). When we go back to our meta-analysis, genes involved in the acquisition of shoot competence should be highly upregulated after 48 h but not yet after 24 h (Che et al., 2007). Only ACR4 and IAA20 fit these requirements (Table 2). ACR4 has been implicated in lateral root initiation (De Smet et al., 2008), but also in shoot morphogenesis and is mainly expressed during embryogenesis in the apical regions (Tanaka et al., 2002). IAA20 is highly upregulated in response to overexpression of ENHANCER OF SHOOT REGENERATION2 (ESR2), which confers cytokinin-independent shoot regeneration to root explants (Ikeda et al., 2006). Based on these characteristics, ACR4 and IAA20 are valuable candidates as markers for the acquisition of shoot competence and their exact role in shoot regeneration should be addressed. Importantly, when the CIM incubation period lasts too long, the organogenesis competence gets irreversibly determined to a root identity and root to shoot conversion is no longer possible (Christianson and Warnick, 1983). Consequently, the optimization of the incubation time on CIM is a crucial step in the establishment of regeneration protocols (Che et al., 2007; Christianson and Warnick, 1983; Valvekens et al., 1988). For example, the Arabidopsis accession C24 has an optimal CIM incubation time of 4 days, after 7 days of treatment, the regeneration efficiency significantly decreases, and after 14 days shoot regeneration no longer occurs (Valvekens et al., 1988). In contrast, the accession Landsberg erecta (Ler) regenerates efficiently even after 14 days of CIM incubation (Gordon et al., 2007). To the best of our knowledge, no genetic or morphological markers have been described that indicate the irreversible commitment of an organ primordium to develop into a root. Several so-called root identity markers, such as WOX5, SCR, SHR, PLT1, RCH1, QC25 or J0121, are also expressed in the premature shoot primordia during regeneration and hence cannot be used to define the root identity (Atta et al., 2008; Sugimoto et al., 2010). Because a lateral root initiation-like process is at the basis of shoot regeneration, it is likely that genuine root identity determining factors only occur in the later stages of lateral root development, such as BDL/IAA12-MP/ARF5 and SHY2/IAA3 (see Section 3.2). However, since BDL/IAA12-MP/ARF5 is also involved in the control of the shoot stem cell niche and the regulation of ESR1 during shoot development (Cole et al., 2009; Zhao et al., 2010), it can be ruled out. By contrast, overexpression of SHY2/IAA3 suppresses axillary shoot meristem formation and expression of the CUC genes (Koyama et al., 2010), which are essential for shoot regeneration (Aida et al., 1999; Daimon et al., 2003; Hibara et al., 2003, 2006; Vroemen et al., 2003). Moreover, SHY2/IAA3 is especially important in the later stages of lateral root formation, since loss of function mutants develop lateral root primordia but fail to emerge the lateral roots, whereas gain of function mutants have less lateral roots due to a reduced number of root primordia (Goh et al., 2012b; Swarup et al., 2008). SHY2/IAA3 also causes cell differentiation in the root meristem through the regulation of PIN expression (Dello Ioio et al., 2008). However, SHY2 expression is observed in leaves and cotyledons as well (Koyama et al., 2010) and is strongly induced by auxin in different tissues (Weijers et al., 2005). So, if SHY2/IAA3 determines root identity, it probably functions locally.



7

Table 2 Genes highly expressed during CIM-incubation (Xu et al., 2012) that are also retained as lateral root initiation genes by Vanneste et al. (2005) and are upregulated in dividing pericycle cells during lateral root initiation (De Smet et al., 2008). Fold change Locus

Name/descriptiona

12 h

24 h

36 h

96 h

At2g46990 At3g59420 At1g80370 At2g26710 At1g68400 At3g57950 At5g18560 At1g08280 At5g05180 At1g62770 At2g14960 At5g06080 At2g45420 At5g10510 At4g00238 At5g05160 At1g55610 At4g24780 At5g39850 At1g73590 At1g28360 At3g15540 At1g15580 At5g51550 At1g64405 At2g39700 At1g69530 At1g03820

IAA20 ARABIDOPSIS CRINCKLE4 (ACR4) CYCA2;4 PHYB ACTIVATION TAGGED SUPPRESSOR1 (BAS1) leucine-rich repeat transmembrane protein kinase unknown protein PUCHI glycosyl transferase family 29 protein unknown protein invertase/pectin methylesterase inhibitor family protein auxin-responsive GH3 family protein LATERAL ORGAN BOUNDARIES DOMAIN PROTEIN33 (LBD33) LATERAL ORGAN BOUNDARIES DOMAIN PROTEIN18 (LBD18) ovule development protein AINTEGUMENTA-LIKE6 (AIL6) REDUCED IN LATERAL GROWTH1 (RUL1) BRASSINOSTEROID INSENSITIVE1 LIKE (BRL1) pectate lyase family protein 40S RIBOSOMAL PROTEIN S9 (RPS9C) PIN1 ETHYLENE RESPONSE FACTOR DOMAIN PROTEIN12 (ERF12) IAA19 IAA5 EXORDIUM LIKE3 (EXL3) unknown protein EXPANSIN4 (EXP4) EXPANSIN1 (EXP1) unknown protein

– – – – – – – – – – – – – 3.57 4.71 6.77 7.63 7.64 7.9 9.07 10.34 10.93 13.57 14.21 20.53 29.01 60.58 217.51

– – 2.81 6.53 7.54 13.04 14.12 14.21 17.06 18.19 27.06 77.92 142.81 4.01 4.94 10.79 9.76 11.42 10.99 13.15 10.43 22.93 13.1 23.77 38.59 25.79 57.89 395.07

13.97 33.71 5.28 12.51 10.07 17.68 20.89 20.85 24.28 17.45 34.03 42.07 124.8 6.07 6.51 9.55 14.2 16.07 8.02 9.7 11.71 29.26 10.81 19.75 22.85 27.66 58.88 445.29

24.15 88.73 10.78 24.54 13.22 17.75 18.76 21.37 37.99 10.98 34.87 – 108.32 10.91 10.94 9.43 20.37 11.45 6.94 12.69 10.34 37.98 – 14.82 11.87 21.85 40.13 180.74

a

Names or descriptions are from The Arabidopsis Information Resource (www.arabidopsis.org).

4. Cytokinin-mediated cell fate respecification and establishment of shoot meristem organization After the auxin-mediated formation of organogenic callus, in the next step of the shoot regeneration process, high cytokinin levels determine the shoot identity of the organ primordia by establishing a shoot stem cell niche (Gordon et al., 2009). Although the available information is at times fragmentary, the success of this process depends on diverse aspects of the overall cytokinin metabolism in plants. These include the uptake and transport of cytokinins, the activity of homeostasis mechanisms, and the induction of cytokinin signaling pathways which will ultimately result in the expression of genes involved in shoot meristem establishment. This last phase in the shoot regeneration process involves auxin-cytokinin crosstalk, redirection of auxin transport, and proper patterning of the expression of key regulatory genes determining the organization of the developing shoot meristem. In the following subsections we will describe the genetic determinants that mediate these different cytokinin-related processes in plants and, when reported, how they affect shoot regeneration. 4.1. Uptake of cytokinins A first prerequisite for shoot formation is that the cytokinins from the SIM reach the cells that have acquired organogenesis competence. Indeed, reduced cytokinin uptake has been reported as a probable cause of recalcitrance (Cortizo et al., 2009). Nevertheless, although the uptake of cytokinins is likely very fast and relatively complete, as suggested by the over 80% removal of cZ or tZ from liquid medium by tobacco BY-2 cells within 15 min (Gajdošová et al., 2011), information on this process during regular plant development is rather scarce. The kinetics of cytokinin uptake are multiphasic and their transport is almost completely abolished by adding a protonophore, therefore, it is assumed that uptake is mainly mediated by multiple proton-coupled cytokinin transport systems (Cedzich et al., 2008). PURINE PERMEASEs (PUP), such as PUP1 and 2, transport free cytokinin bases and, to a

minor extent, also nucleosides, which are precursors of the free cytokinin bases (Bürkle et al., 2003; Gillissen et al., 2000). However, kinetic properties indicate that PUP-mediated transport is inefficient and, thus, other mechanisms likely contribute to the cytokinin transport (Frébort et al., 2011). For instance, EQUILIBRATIVE NUCLEOSIDE TRANSPORTERS (ENTs), such as ENT3 and ENT8 in Arabidopsis and ENT2 in rice, are able to transport nucleosides as well (Hirose et al., 2005; Li et al., 2003; Möhlmann et al., 2001; Sun et al., 2005; Wormit et al., 2004). Because Arabidopsis ent3 or ent8 mutants show a severely reduced uptake of the nucleosides (Sun et al., 2005), it seems that this transport mainly occurs via ENTs and not via uptake by simple diffusion. The effect of pup or ent mutations on shoot regeneration has not been reported, but it would be highly informative to determine the importance of cytokinin transport in this developmental process. 4.2. Cytokinin biosynthesis and metabolism The endogenous cytokinin level and cytokinin homeostasis in plants mainly depend on biosynthesis, degradation and modification (for reviews, see Frébort et al., 2011; Sakakibara, 2006; Spíchal, 2012; Zalabák et al., 2013). Several lines of evidence illustrate that defects in these mechanisms not only disturb the final cytokinin level, but also influence the shoot regeneration capacity (Table 1). Adenosine phosphate-isopentenyltransferases (IPTs) add an isoprenoid chain to the adenine moiety of ADP or ATP and determine the rate-limiting step in cytokinin biosynthesis (Kakimoto, 2001; Takei et al., 2001). Overexpression of IPTs eliminates the requirement of cytokinin in the medium during regeneration and causes spontaneous shoot formation on callus (Kakimoto, 2001; Kunkel et al., 1999; Sun et al., 2003). As such, inducible IPT expression has been used for marker-free transformation of plants (Kunkel et al., 1999; Zuo et al., 2002). Loss of function ipt mutants show a decreased regeneration capacity (Cheng et al., 2013) and have reduced shoot meristem sizes (Miyawaki et al., 2006). The latter can only partially be rescued by cytokinin application, implying that in addition to the cytokinin level, the spatial cytokinin


8


distribution is an important developmental factor. The cytochrome P450 monooxygenases, CYP735A1 and CYP735A2, are specifically involved in trans-zeatin synthesis from 2-iP nucleotides (Takei et al., 2004). Although the knowledge about the role of these CYP proteins in homeostasis is still limited, they may have a positive effect on shoot formation. Indeed, the phenotype of the semi-dominant Arabidopsis mutant uni-1D, that forms shoot meristems on leaves (Igari et al., 2008), was suggested to be caused by induction of CYP735A2 (Uchida et al., 2011). Besides de novo biosynthesis, the cytokinin level can be increased by the conversion of inactive cytokinin nucleotides to the active free bases by LONELY GUY (LOG) enzymes (Kurakawa et al., 2007). Loss of function mutants have similar phenotypes as ipt mutants (Kuroha et al., 2009), including reduced (Tokunaga et al., 2012) or prematurely terminated (Kurakawa et al., 2007) shoot meristems. Overexpressing LOG genes results in cytokinin response phenotypes (Kuroha et al., 2009), but the effect on shoot regeneration has not been explored. An important downregulating mechanism for cytokinin homeostasis is the irreversible degradation by CYTOKININ OXIDASE/DEHYDROGENASE (CKX) enzymes. CKX overexpression leads to a disorganized shoot meristem and sometimes to an arrested shoot growth in an early stage of development (Werner et al., 2003), and also to a reduced regeneration capacity (Yang et al., 2003). Interestingly, the total CKX activity, which is genotype dependent, has been suggested to be an important cause of regeneration recalcitrance (Auer et al., 1992, 1999; Sriskandarajah et al., 2006). As such, this type of recalcitrance can potentially be neutralized by CKX inhibitors or by alternative cytokinins that are more resistant to CKX degradation (Galuszka et al., 2007). The use of CKX inhibitors has indeed proven to be an effective approach to stimulate shoot regeneration (Motte et al., 2013). Since CKX genes are expressed in organ primordia (Werner et al., 2003), CKX inhibitors might locally augment the cytokinin content in regions were shoots can be induced and, as such, be more effective for shoot regeneration than the classical use of traditional cytokinins (Motte et al., 2013). Finally, the active cytokinin content is also reduced by the inactivating effect of glucosylation. In Arabidopsis, the glucosyltransferases UGT76C1 and UGT76C2 are able to irreversibly glucosylate cytokinins at the N7 and N9 positions (Hou et al., 2004). UGT76C2 was demonstrated to be involved in cytokinin homeostasis, but loss and gain of function mutants did not cause any obvious phenotypes (Wang et al., 2011a). On the other hand, O-glucosylation of zeatin is reversible, and mainly depends on UGT85A1 (Hou et al., 2004; Jin et al., 2013). These conjugated forms are resistant to CKX degradation (Galuszka et al., 2007) and are suggested to be storage forms (Mok and Mok, 2001). Therefore, together with the relatively slow effect of IPT-mediated de novo biosynthesis (Kakimoto, 2001; Takei et al., 2001), the conversion of O-glucosylated cytokinins is thought to be important for rapidly attaining cytokinin homeostasis (Frébort et al., 2011; Jin et al., 2013). The O-glucosyl derivatives are cleaved by specific β-glucosidases (Brzobohaty et al., 1993) and overexpression of the maize β-GLUCOSIDASE1 (ZmGLU1) in Arabidopsis results in a higher regeneration capacity (Klemš et al., 2011). Because genes encoding such β-glucosidases have only been identified in Zea mays and Brassica napus (Brzobohaty et al., 1993; Falk and Rask, 1995), it is currently not known how common this particular homeostasis mechanism is in plants. Although GLYCOSIDE HYDROLASE (GH) FAMILY 1 β-GLYCOSIDASE19 (BGLU19) from Arabidopsis has been proposed to encode a β-glucosidase (Xu et al., 2004), closely related proteins appear to have different substrates (Zhao et al., 2012), casting doubt on the ability of BGLU19 to cleave O-glucosylated cytokinins. Hence, as there is no biochemical evidence supporting the occurrence of this metabolic step in Arabidopsis, the general fate of O-glucosylated cytokinins remains unclear. 4.3. Cytokinin signaling In Arabidopsis, three hybrid histidine kinases, AHK2, AHK3 and AHK4/WOODENLEG (WOL)/CYTOKININ RESPONSE1 (CRE1), perceive

cytokinins each with their own ligand binding activity and specificity (Inoue et al., 2001; Spíchal et al., 2004; Suzuki et al., 2001; Ueguchi et al., 2001; Yamada et al., 2001). Loss of function mutations in AHK2 and AHK3 result in a reduced regeneration capacity and the lack of a functional AHK4 leads to a complete recalcitrance that cannot be rescued by elevated cytokinin concentrations in the SIM (Higuchi et al., 2004; Inoue et al., 2001; Motte et al., 2013; Nishimura et al., 2004; Ueguchi et al., 2001). Possibly, recalcitrance can originate from the inability of the cytokinin receptors of the plant to efficiently bind to the cytokinin used in the SIM (Spíchal et al., 2004). If so, this type of recalcitrance could perhaps be rescued by using other cytokinins. Whereas in intact plants, AHK4 is predominantly expressed in the vascular bundle and the pericycle of the root (Higuchi et al., 2004; Mähönen et al., 2000), during CIM incubation it accumulates at particular sites increasing their cytokinin sensitivity and predisposing them for shoot formation (Gordon et al., 2009). Overexpression of CYTOKININ INDEPENDENT KINASE (CKI1), that shares the kinase domain with the AHKs, increases cytokinin signaling, resulting in cytokinin-independent shoot regeneration (Hwang and Sheen, 2001). Upon cytokinin binding, the kinase activity of the AHKs is triggered, resulting in the phosphorylation of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEINs (AHPs). These AHPs serve as shuttles to transfer phosphoryl groups to ARABIDOPSIS RESPONSE REGULATORs (ARRs) (Hwang and Sheen, 2001; Hwang et al., 2012; Imamura et al., 1999) and are important for the movement of CYTOKININ RESPONSE FACTORs (CRFs) into the nucleus (Cutcliffe et al., 2011; Hwang et al., 2012; Rashotte et al., 2006). There, type B ARRs, activated by phosphorylation, and CRFs induce transcription of cytokinin-controlled genes and type A ARRs, which act in a negative feedback loop of cytokinin signaling (Cutcliffe et al., 2011; Hwang and Sheen, 2001; Hwang et al., 2012; Imamura et al., 1999; Rashotte et al., 2006). Overexpression or elimination of AHP1-5 induces only moderate cytokinin-like effects due to functional redundancy (Hutchison et al., 2006). AHP6, which is unable to receive a phosphoryl group, is suggested to interact with the phosphorelay machinery and hence inhibits cytokinin signaling (Mähönen et al., 2006). The stimulation or inhibition of cytokinin signaling downstream of the AHPs dramatically affects shoot regeneration. For example, loss of function mutations in type B ARRs, which activate the cytokinin response, or in particular CRFs, reduce the regeneration capacity (Ishida et al., 2008; Rashotte et al., 2006). In contrast, overexpression of type B ARR2 and ARR11 results in cytokinin-independent shoot regeneration (Hwang and Sheen, 2001) and in spontaneous outgrowth of adventitious shoots on the junction of cotyledons and of leaf and petioles, respectively (Imamura et al., 2003). Ectopic expression of these Arabidopsis transcription factors increases regeneration in other plants as well, such as tobacco (Rashid and Kyo, 2010). Interestingly, expression profiling of a group of regeneration recalcitrant Arabidopsis lines within an inbred population revealed that the expression level of the type B ARR18 was much lower than in the highly regenerative lines (Lall et al., 2004), implying that the level of type B ARR expression is one parameter that determines regeneration capacity. Because the primary response type A ARRs mediate a negative feedback on cytokinin signaling (Hwang and Sheen, 2001), overexpression of for instance ARR7 or ARR15 reduces cytokinin signaling and decreases regeneration capacity (Buechel et al., 2010; Kiba et al., 2003). Loss of function mutants of ARR7 and ARR15 on the other hand, show an increased regeneration capacity (Buechel et al., 2010). Finally, the type C ARRs, ARR22 and ARR24, which are not induced by cytokinins, also block cytokinin signaling (Gupta and Rashotte, 2012; Kiba et al., 2004). Type C ARRs act as phosphatases that receive phosphoryl groups and reduce cytokinin signaling. They are presumably very important for the local regulation of cytokinin signaling, because even a slight misexpression severely interferes with cytokinin homeostasis (Horak et al., 2008). Consequently, for instance, ARR22



overexpression results in complete regeneration recalcitrance (Kiba et al., 2004).

4.4. Cytokinin-induced WUSCHEL expression marks the onset of shoot meristem formation During regular plant development, all above-ground tissues originate from the shoot apical meristem (SAM). The SAM is organized in a central zone at the tip, a rib meristem underneath, and the peripheral zone where leaf and flower primordia originate. The central zone harbors pluripotent stem cells, whose daughter cells are displaced into the rib meristem and peripheral zone and differentiate into cells required for the formation of the stem or the lateral organs, respectively (for reviews, see Barton, 2010; Dodsworth, 2009; Perales and Reddy, 2012). This organization of the SAM and the stem cells has to be maintained to assure indeterminate growth of the plant. An important mechanism in the stem cell maintenance is the WUS/CLV circuit (Schoof et al., 2000). WUS encodes a transcription factor that regulates the transcription of numerous genes involved in meristem-related processes and cell division (Busch et al., 2010). In response to cytokinins, it is expressed in the organizing center of the central zone of the SAM (Gordon et al., 2009; Mayer et al., 1998). WUS migrates to the overlaying stem cells where it directly induces CLAVATA3 (CLV3) expression (Brand et al., 2000; Mayer et al., 1998; Yadav et al., 2011). CLV3 encodes a signaling peptide which restricts WUS expression to the cells of the organizing center (Brand et al., 2000; Lenhard and Laux, 2003) via a mechanism that is not completely understood. The CLV3 signal transduction includes at least proteolysis of the CLV3 peptide, probably into a 12-amino acid peptide (MCLV3) (Kondo et al., 2006; Ni and Clark, 2006), activation of different receptor kinase complexes, such as CLV1 and RECEPTOR-LIKE PROTEIN KINASE2 (RPK2) homomers, and CLV1BARELY ANY MERISTEM1 (BAM1), CLV1-BAM2 and CLV2-CORYNE (CRN)/SUPPRESSOR OF LLP1 2 (SOL2) heteromers (Clark et al., 1997; DeYoung and Clark, 2008; Guo et al., 2010; Kayes and Clark, 1998; Kinoshita et al., 2010; Miwa et al., 2008; Müller et al., 2008), and inhibition of the phosphatases POLTERGEIST (POL) and POL-LIKE1 (PLL1) (Gagne and Clark, 2010; Song et al., 2006). The role of the multiple receptor kinase complexes is currently unclear, but CLV1 and CLV2CRN seem to be most important in the shoot CLV3 signal transduction, since clv1, clv2 and crn mutants have severe shoot development phenotypes (Miwa et al., 2008; Müller et al., 2008). During shoot regeneration, incubation on SIM leads to the expression of multiple cytokinin-regulated genes that are essential for shoot initiation (Che et al., 2002). As such, the cytokinin signaling pathway also induces WUS (Gordon et al., 2009). As a transcription factor, WUS directly represses the expression of the type A ARRs (Leibfried et al., 2005), which affects cytokinin signaling and hence shoot regeneration (Buechel et al., 2010). The central position of the WUS-CLV mechanism in proper organization of the shoot meristem is also of utmost importance for shoot regeneration. During SIM incubation WUS is expressed in two phases (Fig. 1). Initially, expression is observed throughout the explant surrounding shoot precursor areas that express CUC2 (see below). In this phase, WUS might have a role in cell respecification (Gordon et al., 2007). Next, WUS expression becomes restricted to regions where the AKH4 cytokinin receptor had been induced during prior CIM incubation; in these regions, a strong cytokinin response is detected and shoots are likely to develop (Cheng et al., 2013; Gordon et al., 2009). Shortly after this WUS expression, CLV3 is expressed in the apex of organ primordia during their conversion to shoot meristems (Chatfield et al., 2013). Loss of function wus mutants show severely reduced or no shoot regeneration (Chatfield et al., 2013; Gordon et al., 2007). Overexpression of WUS, on the other hand, causes improved shoot regeneration (Gallois et al., 2004). As expected, also mutants in target genes of WUS show an altered regeneration capacity (Chatfield et al., 2013).

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4.5. Auxin-cytokinin crosstalk and auxin transport are important factors controlling shoot meristem organization The cytokinins present in SIM are determinative for the conversion of the organ primordia into shoot meristems. Nonetheless, the success of shoot regeneration at this stage is also controlled by auxins and the auxin-cytokinin crosstalk (Su et al., 2011). For instance, the PIN auxin efflux carrier genes are upregulated during SIM incubation (Atta et al., 2008; Gordon et al., 2007), specifically at those sites that showed increased expression of AHK4 during incubation on CIM (Gordon et al., 2009). A similar auxin-cytokinin crosstalk has been reported during root formation and growth, where cytokinins were shown to regulate PIN expression (Marhavý et al., 2011; Pernisová et al., 2009; Růžička et al., 2009). In addition, cytokinins induce auxin biosynthesis both in young shoot tissue (Jones et al., 2010) and during shoot regeneration (Cheng et al., 2013), further contributing to the establishment of the auxin gradient. Conversely, auxin controls the cytokinin levels by suppressing the expression of the shoot meristem gene SHOOT MERISTEMLESS (STM), which promotes cytokinin biosynthesis (Heisler et al., 2005; Yanai et al., 2005). Auxin also influences cytokinin distribution by negative regulation of IPTs, involving the auxin response factors ARF3 and MP/ARF5 and the A-type ARRs ARR7 and ARR15 (Cheng et al., 2013; Zhao et al., 2010). During embryogenic shoot development, the cytokinin-induced WUS activates TOPLESS (TPL) (Kieffer et al., 2006), which reduces auxin signaling via interaction with IAA12/BDL and MP/ARF5 (Long et al., 2006; Szemenyei et al., 2008). Other auxin response factors, such as ARF10, ARF16 and ARF17, are possibly involved in the cytokinin-auxin cross-talk as well. Indeed, the microRNA gene MIR160a, which negatively regulates the expression of these ARFs, is specifically down-regulated after 10 days of SIM incubation and overexpression of MIR160a strongly reduces the regeneration capacity (Qiao et al., 2012a). Moreover, transgenic lines with a non-degradable form of ARF10 exhibit an increased expression of WUS, resulting in a stronger shoot regeneration capacity, while loss of function decreases the regeneration capacity (Qiao and Xiang, 2013; Qiao et al., 2012a). Interestingly, in contrast to other ARFs, the MIR160 regulated ARFs have a negative effect on lateral root initiation (Mallory et al., 2005; Wang et al., 2005), substantiating their particular involvement in the cytokinindependent part of shoot regeneration. After all, it is likely that several auxin-related key players discussed in Section 3, might affect shoot regeneration both during CIM- and SIM-related events. Prior to shoot formation on SIM, the epidermal cells of CUC2-marked shoot precursors are enriched in auxin-transporting PIN1 proteins that are directed towards the apical tip (Fig. 1). Hence, auxin maxima are observed at the tip of these premature organ primordia (Atta et al., 2008; Gordon et al., 2007). In this phase, WUS expression is still dispersed and surrounds the CUC2-marked areas (Gordon et al., 2007). In a subsequent phase, during the actual meristem morphogenesis, PIN1 expression shifts towards incipient organ primordium sites, creating auxin maxima essential for organ formation and the phyllotactic patterning of the new meristem (Fig. 1; Gordon et al., 2007; Heisler et al., 2005). PIN1 expression is functionally required for efficient shoot regeneration, since shoot formation is severely reduced in pin1 mutants (Gordon et al., 2007). Because shoot regeneration at this stage can be completely blocked by application of auxin transport inhibitors, it seems that correct PIN1 localization is equally important (Cheng et al., 2013; Christianson and Warnick, 1983). While PIN1 transports auxin towards the shoot tip or the incipient organ primordium sites (Gordon et al., 2007), the auxin influx carrier AUX1, which is functionally redundant with LAX1-3 (Bainbridge et al., 2008), is assumed to be involved in restricting auxin to the epidermal cell layer (Reinhardt et al., 2003; Vernoux et al., 2010). The aux1 lax1 lax2 lax3 quadruple mutant does not show alterations in shoot meristem structure, but it is defective in the subsequent phyllotactic patterning (Bainbridge et al., 2008). Hence, in the latest phase of shoot regeneration, these


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auxin influx carriers might be important after the PIN1 polarization, for the patterning of the leaf primordia in the developing shoot meristem. During shoot, root and embryo development, shifts of PIN1 polarization are mediated by PINOID (PID) (Benjamins et al., 2001; Friml et al., 2004; Furutani et al., 2004). This protein kinase might have the same role during shoot regeneration. Indeed, PID is upregulated especially in presumptive shoot sites during SIM incubation and pid loss of function causes a decreased shoot regeneration capacity (Matsuo and Banno, 2012). Shoot-related auxin-transport is additionally regulated by ESR1/DÖRNROSCHEN (DRN) and ESR2/DRN-LIKE (DRNL), two partially redundant AP2 transcription factors that interact during shoot development with PIN1 and PID, respectively (Chandler et al., 2011a). ESR1 was first discovered in a cDNA overexpressor screen for cytokininindependent shoot regeneration, but overexpression also greatly increased shoot regeneration efficiency in the presence of cytokinin. ESR1 is rapidly induced during SIM incubation and its expression is dependent on CIM pre-treatment (Banno et al., 2001). Overexpression of ESR2, which is highly similar to ESR1, causes a comparable regeneration phenotype, but it is expressed later during SIM incubation (Ikeda et al., 2006; Matsuo et al., 2009). Based on this temporal difference in expression, it has been suggested that ESR1 is mainly important during the conversion of the early lateral root primoridum into a shoot meristem, while ESR2 functions during the subsequent shoot development (Matsuo and Banno, 2012). Moreover, the observations that ESR2 expression precedes the establishment of auxin maxima in incipient primordia during embryo development (Chandler et al., 2011b) and that esr knockouts are partially regeneration recalcitrant (Matsuo et al., 2011), corroborates the role of the ESR genes in shoot regeneration. 4.6. CUC-STM interplay during shoot initiation and development The two phases in auxin transport during shoot regeneration concur with two distinct phases of CUC2-expression (Fig. 1). In the first phase CUC2 is expressed throughout the primordia, while in the second phase, it is restricted to the peripheral zone of the SAM (Gordon et al., 2007). In embryo development, the homologous CUC1 and CUC3 are partially redundant to CUC2 (Aida et al., 1997, 1999; Vroemen et al., 2003), but while CUC1 has a comparable yet slightly delayed expression pattern to CUC2 during shoot regeneration (Cary et al., 2002), the expression of CUC3 is not notably modulated during CIM or SIM incubation (Che et al., 2006). Expression of CUC1 and CUC2 during shoot regeneration activates STM (Daimon et al., 2003; Hibara et al., 2003). STM is a transcription factor belonging to the class 1 KNOTTED-1-like HOMEOBOX family that is essential for shoot meristem initiation and for the maintenance of the undifferentiated cells in the central zone of the SAM (Barton and Poethig, 1993; Endrizzi et al., 1996; Long et al., 1996). STM is first expressed in a ring of cells surrounding CUC2expressing primordia and afterwards, when the relocalization of PIN1 during shoot meristem morphogenesis has occurred, throughout the entire meristem (Fig. 1; Gordon et al., 2007). In the latter stage, STM is required to restrict the expression of the CUC genes to the peripheral zone of the SAM (Aida et al., 1999; Cary et al., 2002; Gordon et al., 2007; Spinelli et al., 2011; Takada et al., 2001; Vroemen et al., 2003). Loss of function mutants in STM are completely recalcitrant, while mutants in the CUC genes exhibit reduced shoot regeneration (Aida et al., 1997; Daimon et al., 2003). Although overexpression of CUC1 or CUC2 increases the regeneration capacity in a wild-type background, it is not sufficient to alleviate the regeneration defect of stm mutants (Daimon et al., 2003). CUC1 activates, dependent upon CUC2, the transcription factors LIGHT-DEPENDENT SHORT HYPOCOTYLS3 (LSH3)/ORGAN BOUNDARY1 (OBO1) and LSH4/OBO4 (Takeda et al., 2011). During the early phases of shoot regeneration, LSH4 seems to have a comparable, but slightly delayed expression pattern as CUC1 (Cary et al., 2002) and during further shoot development, LSH3 and LSH4 expression resembles

that of the CUC genes (Cary et al., 2002; Cho and Zambryski, 2011; Takeda et al., 2011). Their exact role in shoot formation or a possible interaction with STM is not known, but LSH3 and LSH4 are important for meristem maintenance and organ differentiation in the peripheral zone of the SAM (Cho and Zambryski, 2011; Takeda et al., 2011). Moreover, LSH3 and LSH4 overexpression induces the formation of WUS-expressing meristem-like tissue, shoot-like primordia expressing WUS and STM, and shoots on flowers (Takeda et al., 2011), and LSH4 is a valuable marker for shoot regeneration (Motte et al., 2013). 4.7. WUS, STM and shoot determination The transcription factors WUS and STM are crucial during meristem formation and maintenance, but they act independently (Lenhard et al., 2002). Indeed, reporter analysis during shoot regeneration revealed no specific order in their expression (Atta et al., 2008). Combined overexpression of WUS and STM causes ectopic shoot formation, while overexpression of only one of the genes does not (Gallois et al., 2002; Lenhard et al., 2002). Hence, WUS and STM together are required and sufficient for de novo shoot formation. Therefore, the moment when WUS and STM expression is initiated just after the PIN1 shift, might mark organ determination and assign shoot identity. Indeed, at least for STM, the timing of expression coincides with the timing that explants can be transferred from SIM to hormone free medium, without affecting the regeneration capacity (Zhao et al., 2002). In addition, the few shoots that regenerate on root explants of a partially dysfunctional wus mutant, have an autonomous development, once STM expression is activated (Gordon et al., 2007). Moreover, Brand et al. (2002) showed, in contrast to other reports, ectopic formation of shoot meristems by STM overexpression, even on defective wus mutants. Hence, it seems that STM, at least when expressed within the meristem, assigns shoot determination. 5. Perspectives With this review we provide but a glimpse of the myriad of factors that determine regeneration capacity. Indeed, alternative regeneration protocols, that do not strictly follow the phases described for the twostep procedure, can be interesting for comparative studies. For instance, regeneration can be accomplished using a single auxin- and cytokininrich medium that combines both phases at the same time. Under these conditions, shoots are often induced from regions with a high auxin level or with potential for callus formation, such as root tips or wounded tissues (eg. Geier, 1986; Haque et al., 1997; Kelkar and Krishnamurthy, 1998). Moreover, hormones, such as ethylene (Chatfield and Raizada, 2008), gibberellins (Jasinski et al., 2005), and regulatory proteins, such as class III type HD-ZIP transcription factors (Catterou et al., 2002; Duclercq et al., 2011a; Gardiner et al., 2011; Green et al., 2005) or cyclin-dependent kinases (Andersen et al., 2008; Meng et al., 2010) also affect shoot regeneration. Additionally, new key factors implicated in shoot regeneration are continuously identified, but their exact role in the process remains to be determined. Examples are photoreceptors, such as PHYTOCHROME (PHY) and CRYPTOCHROME (CRY), and interacting proteins, including ELONGATED HYPOCOTYL5 (HY5) (Nameth et al., 2013), the transcription factor RAP2.6L (Che et al., 2006), the fasciclin-like arabinogalactan-protein FLA1 (Johnson et al., 2011), and the oxygen-binding hemoglobins (Wang et al., 2011b). The recent report of Iwase et al. (2011), concerning auxin independent callus formation induced by the transcription factor WOUND INDUCED DEDIFFERENTIATION1 (WIND1), might be of major importance for regeneration research as well. The WIND1-induced callus does not show a lateral root-like gene expression program and is not disturbed in the slr mutant (Iwase et al., 2011). Hence, WIND1dependent shoot regeneration follows a different pathway, opening novel opportunities to solve regeneration recalcitrance by using this different type of callus.



Although we mainly discussed data from Arabidopsis, insights gained from this model organism are likely to be useful for solving regeneration problems in plant tissue culture in general, because shoot organogenesis in Arabidopsis and in other plants appears to exhibit strong anatomical and hence possibly also molecular similarities. For instance, the emergence of shoots at sites opposite the protoxylem poles of the pericycle (Beijerinck, 1887; Che et al., 2007) and the resemblances in lateral root and adventitious shoot initiation seem to be conserved themes (Atta et al., 2008; Bonnett and Torrey, 1966; Peterson, 1970; Spencer-Barreto and Duhoux, 1994). Moreover, analysis of auxin- and cytokinin-related processes, which are critically important for shoot organogenesis and highly conserved in higher plants (De Smet et al., 2011; Finet and Jaillais, 2012; Spíchal, 2012), have uncovered several causes of recalcitrance not only in Arabidopsis (for examples, see Pal et al., 2012; Sriskandarajah et al., 2006). Altogether, by focusing on the most important events that occur in the two-step regeneration protocol during incubation on auxin-rich CIM and cytokinin-rich SIM, we could identify hinge points and novel candidate genes for controlling shoot regeneration. These factors merit further study both in Arabidopsis and other plants, because they can lead to novel insights in the shoot regeneration process and possibly shed light on the molecular basis of regeneration capacity or recalcitrance. The control of these factors might provide breakthroughs in solving shoot regeneration problems and, more importantly, open the path for in vitro propagation and genetic engineering of recalcitrant plants. For instance, to understand the cytokinin-mediated steps in shoot regeneration we used a chemical screen approach combined with LSH4 as a marker and identified phenyl-adenine as a CKX inhibitor that significantly improved shoot induction (Motte et al., 2013). Similarly, Auer et al. (1992; 1999) and Sriskandarajah et al. (2006) discovered that certain recalcitrant Petunia or cacti cultivars had excessive CKX activity, and Pal et al. (2012) found that recalcitrant potato cultivars exhibited a high endogenous auxin level, all identifying possible causes for regeneration recalcitrance and providing means to overcome these problems. These examples show that progress made in basic research on shoot regeneration can genuinely benefit tissue culture practices and, therefore, it should be implemented much more intensively during the establishment of working protocols for recalcitrant plants. As such, by studying diverse cultivars and explants one might get insight into the basis of recalcitrance of the plant of interest and step away from pure trial and error towards approaches inspired by more educated guesses to solve tissue culture problems. Acknowledgments We thank Tom Beeckman and two anonymous reviewers for their critical reading of the manuscript and valuable contributions. References Ahmad P, Ashraf M, Younis M, Hu X, Kumar A, Akram NA, et al. Role of transgenic plants in agriculture and biopharming. Biotechnol Adv 2012;30:524–40. Aida M, Ishida T, Fukaki H, Fujisawa H, Tasaka M. Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant. Plant Cell 1997;9: 841–57. Aida M, Ishida T, Tasaka M. Shoot apical meristem and cotyledon formation during Arabidopsis embryogenesis: interaction among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS genes. Development 1999;126:1563–70. Andersen SU, Buechel S, Zhao Z, Ljung K, Novák O, Busch W, et al. Requirement of B2-type cyclin-dependent kinases for meristem integrity in Arabidopsis thaliana. Plant Cell 2008;20:88–100. Atta R, Laurens L, Boucheron-Dubuisson E, Guivarc'h A, Carnero E, Giraudat-Pautot V, et al. Pluripotency of Arabidopsis xylem pericycle underlies shoot regeneration from root and hypocotyl explants grown in vitro. Plant J 2008;57:626–44. Attfield EM, Evans PK. Stages in the initiation of root and shoot organogenesis in cultured leaf explants of Nicotiana tabacum cv. Xanthi nc. J Exp Bot 1991;42:59–63. Auer CA, Cohen JD, Laloue M, Cooke TJ. Comparison of benzyl adenine metabolism in 2 Petunia-hybrida lines differing in shoot organogenesis. Plant Physiol 1992;98: 1035–41.

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Shoot regeneration in root cultures of Solanaceae.

166 in Arabidopsis thaliana.

Efficient shoot regeneration of Brassica campestris using cotyledon explants cultured in vitro.

An efficient in vitro plantlet regeneration from shoot tip cultures of Curculigo latifolia, a medicinal plant.

High frequency adventitious shoot regeneration from excised leaves ofPaulownia spp. cultured in vitro.

Factors influencing in vitro shoot regeneration from leaf segments of Chrysanthemum.

Adventitious shoot regeneration from leaf tissue of three pear (Pyrus sp.) cultivars in vitro.

Genetic control of in vitro shoot regeneration from leaf explants inSolanum chacoense Bitt.

In vitro shoot regeneration from leaf mesophyll protoplasts of hybrid poplar (Populus nigra x P. maximowiczii).

In vitro propagation of Achillea asplenifolia VENT. through multiple shoot regeneration.

A collective path toward regeneration.

Shoot regeneration from Glycine canescens tissue cultures.

Molecular approaches to nerve regeneration.

Molecular insights provide the critical path to disease mitigation.

Microfilament Depolymerization Is a Pre-requisite for Stem Cell Formation During In vitro Shoot Regeneration in Arabidopsis.

Rapid transformation ofMedicago truncatula: regeneration via shoot organogenesis.

Predictive correlates of shoot regeneration from potato protoplast culture.

Plant regeneration from shoot callus of rosewood (Dalbergia latifolia Roxb.).

Callus induction and regeneration via shoot tips of Dendrocalamus hamiltonii.

Studies on shoot regeneration of lupins (Lupinus spp.).

Shoot regeneration from stem and leaf callus of Eucalyptus tereticornis.

In vitro propagation of taro, with spermine, arginine, and ornithine : I. Plantlet regeneration from primary shoot apices and axillary buds.

Enhanced shoot regeneration from Brassica campestris by silver nitrate.

De novo shoot organogenesis of Pinus eldarica Medw. in vitro : I. Reproducible regeneration from long-term callus cultures.