Introduction

Historically, the study of plant cell growth and development focused on the role of phytohormones (Darwin 1880; Skoog and Miller 1957). However, at the end of twentieth century, with the advent of molecular genetics and transcriptomics, plant growth and development became more focused on various transcription factors (TFs) and their diverse signaling pathways as well as their expression patterns. Several studies at the molecular level have suggested that plant cell dedifferentiation mainly depends on the sequential and proper expression of a number of TF genes which are required for morphogenesis (Zuo et al. 2002; Bouchabké-Coussa et al. 2013; Zhai et al. 2016; Rupps et al. 2016; Songstad et al. 2017; Jha and Kumar 2018; Kumar and Van Staden 2019; Gordon-Kamm et al. 2019; Kausch et al. 2019). More specifically, during embryogenesis, many TFs are involved in vegetative to embryo transition such as SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KNASE (SERK) (Schmidt et al. 1997), WUSCHEL (WUS) (Zuo et al. 2002), BABY BOOM (BBM) (Boutilier et al. 2002), LEAFY COTYLEDON1/2 (LEC1/2) (Harada 2001), FUSCA3 (FUS3) (Luerûen et al. 1998), EMBRYO MAKER (Tsuwamoto et al. 2010), ABAINSENSITIVE3 (ABI3) (Shiota et al. 1998), and AGAMOUS-LIKE15 (AGL15) (Harding et al. 2003). Among the different TFs which participate in the expression and regulation of SE, WUS gene is reported to play an essential role (Laux et al. 1996; Zuo et al. 2002; Meng et al. 2019).

WUSCHEL (WUS) gene encodes the homeodomain TF, originally identified as a master regulator required for shoot and floral meristem integrity in Arabidopsis (Laux et al. 1996; Mayer et al. 1998). Ectopic overexpression of WUS gene regulates cell fate during cell dedifferentiation including size of shoot meristem, somatic embryo, adventitious shoot and lateral leaf formation, by maintaining the pluripotent stem cells (Zuo et al. 2002; Gallois et al. 2004; Honda et al. 2018) (Fig. 1).

Fig. 1
figure 1

A schematic model of the WUSCHEL (WUS) transcription factor gene showing the regulation of diverse functions in plant growth and development. As shown in Fig. 1, the stem cells are maintained in the apical shoot meristem by a regulatory feedback loop between the stem cells and organizing center (OC), via CLV3 and WUS expression. The shoot apical meristem (SAM) organization with the central zone (CZ), peripheral zone (PZ) and rib zone (RZ) is shown. The stem cells (shown in red) express the CLV3 signaling and OC (shown in green) induces the WUS expression. The WUS TF gene binds with ARABIDOPSIS RESPONSE REGULATOR7 (ARR7) gene (Type-A response regulator which negatively regulates cytokinin signaling) to suppress its expression (To et al. 2004, 2007; Leibfried et al. 2005). However, the ARR7/15 expression is regulated by AUXIN RESPONSE FACTOR5/MONOPTEROS activation (Zhao et al. 2010). The expression of WUS and ARRs is positively regulated by cytokinin signaling (Holt et al. 2014). On the other hand, during SE, the WUS TF gene transcriptionally regulates LEC1, LEC2 and AGL15 genes. The LEC1 gene expressed the YUC gene (encodes a biosynthesis enzyme), whereas LEC2 and AGL15 activate the expression of TAA1 (encodes an auxin biosynthesis enzyme) and IAA30 (negative regulator of auxin) (Horstman et al. 2017). In addition, AGL15 positively regulates Gibberellin (GA) degrading enzyme GA2ox6, and negatively regulates the biosynthesis gene GA3ox2, resulting in a reduced endogenous GA level (Ikeuchi et al. 2015).The WOX gene shows multiple functions including peptide signaling (Li et al. 2018), stem cell regulation (Dolzblasz et al., 2016), early and lateral leaf development (Honda et al. 2018; Yasui et al. 2018) and embryo development (Palovaara et al. 2010; Tvorogova et al. 2019) during plant growth development. In addition, BBM TF gene also binds to YUC and TAA1 gene (Horstman et al. 2017). Arrows with a solid line show direct regulation and arrows with dotted lines indicate indirect regulation whose mechanisms are not clear as yet. CZ central zone, GA gibberellin, PZ peripheral zone, RZ rib zone, OC organizing center

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The expression of WUS TF gene is confined to a small group of cells in the central zone of the shoot apical meristem (SAM) and is required for the maintenance of stem cell fate in Arabidopsis (Mayer et al. 1998; Yadav et al. 2011). The SAM organization in Arabidopsis includes three different layers of stem cells (L1-L3), a central zone (CZ), a peripheral zone (PZ) and a rib zone (RZ). WUS gene is expressed in the organizing centre (OC) and promotes the expression of CLAVATA3 (CLV3) in the stem cells (Schoof et al. 2000). Several reports reveal that the WUS TF gene can act both as a transcriptional repressor of cytokinin response genes and a transcriptional activator of the floral gene AGAMOUS, suggesting that its molecular mechanism is modified dependent on the developmental context (Lohmann et al. 2001; Leibfried et al. 2005; Kieffer et al. 2006; Ikeda et al. 2009). Likewise, WUS gene also acts as a positive regulator for the expression of CLV3, which negatively regulates the meristem size by suppressing WUS expression (Schoof et al. 2000; Reddy 2008; Ikeda and Ohme-Takagi 2014), although WUS gene product mainly acts as a transcriptional repressor to suppress CLV3 expression (Ikeda et al. 2009).

In vitro plants can be regenerated via organogenesis or embryogenesis (Su and Zhang 2014; Gaillochet and Lohmann 2015; Kumar and Van Staden 2017), and WUS is the only TF that has been shown to be involved in regulation of both embryogenic (totipotent) and meristematic (pluripotent) stem cells to date. Although significant progress has been made in knowledge of WUS TF gene to understand its signaling pathway, the molecular mechanisms underlying embryo development and stem cell fate development still remain unclear. This review provides insights on the recent discoveries and state-of-the-art advances on WUS TF gene in the area of plant growth and development.

WUS-mediated regulation of the shoot meristem

The homeobox gene WUS, which is differentially expressed in the OC of the SAM, is required for stem cell identity and plays an important role in the regulation of shoot meristem (Laux et al. 1996; Mayer et al. 1998; Schoof et al. 2000). WUS is synthesized in the OC, then migrates into the CZ to directly activate CLV3 by binding to its promoters (Yadav et al. 2011), and further WUS-CLV interactions create a feedback circuit between the OC and the stem cells to establish the shoot stem cell niche (Schoof et al. 2000; Zhou et al. 2015; Galli and Gallavotti 2016), as illustrated in Fig. 1. In this context, WUSCHEL-RELATED HOMEOBOX (WOX), a homologous of WUS gene, and HAIRY MERISTEM (HAM) TFs are transcriptionally downregulated via CLV3 signal into the nucleus, while WUS interacts with HAM1 and HAM2 to regulate target gene expression and maintenance of stem cells (Zhou et al. 2015). Parallel to this, WOX4 interacts with HAM4 and is expressed in Arabidopsis procambial cells (Ji et al. 2010), while WOX5 interacts with HAM2, it is expressed in the root apical meristem (RAM) and is involved in the control and maintenance of shoot and root stem cell niche (Sarkar et al. 2007; Zhou et al. 2015; Somssich et al. 2016).

A recent study in Medicago truncatula showed that HEADLESS (HDL), a homolog of AtWUS, is essential for shoot meristem regulation and leaf development (Meng et al. 2019), as HDL interacts with the transcriptional co-repressor MtTPL (TOPLESS) and acts as a transcriptional repressor in shoot meristem maintenance. Likewise, several other studies also suggested that WUS acts as a transcriptional repressor in the maintenance of shoot meristem through its interaction with TPL and TPL-related transcriptional co-repressor, which are employed by their conserved WUS-box (Ikeda et al. 2009; Dolzblasz et al. 2016; Meng et al. 2019). Furthermore, in their work using genetic analysis, Meng et al. (2019) also found that HDL and STENOFOLIA (STF), a master regulator of M. truncatula lamina outgrowth, regulate leaf blade development. Interestingly, the leaf blade outgrowth regulation by STF had been previously reported (Tadege et al. 2011; Lin et al. 2013; Zhang et al. 2014), implying that WUS would also be involved in the regulation of leaf blade growth.

As far as the relationship of WUS with auxins is concerned, in A. thaliana, WUS was shown to act rheostatically and restrict auxin signaling pathway to maintain the stem cell identity (Ma et al. 2019). This rheostatic activity of WUS is hypothesized to occur via regulation of histone acetylation and interference with HISTONE DEACETYLASES (HDAC) activity, which triggers auxin pathway in stem cells (Zhou et al. 2018). Loss of WUS action in the axillary meristem and SAM reduce shoot development, even if WUS overexpression encourages ectopic shoot growth development (Wang et al. 2017).

WUS involvement in floral and reproductive organ development

The regulation of WUS expression also involves an epigenetic mechanism network in the context of floral meristem development (Cao et al. 2015). In this context, Sun et al. (2019) recently showed that WUS is repressed by KNUCKLES (KNU) through histone deacetylation in the floral meristem. Bimolecular fluorescence complementation (BiFC) assays indicated that KNU physically interacts at the nucleus with FERTILIZATION INDEPENDENT ENDOSPERM (FIE), a Polycomb Repressive Complex2 (PRC2) component, and thereby mediates the subsequent deposition of the epigenetic repression via histone H3K37me3 for the stable silencing of WUS (Sun et al. 2019).

In earlier work, in situ hybridization revealed that WUS is expressed in immature stomium cells and is involved in the anther development. As a result of this interaction, anthers of wus mutants had less and malformed lobes compared to the wild type, showing that WUS is essential for normal anther development (Deyhle et al. 2007). A recent study in Chrysanthemum morifolium also indicated that WUS interacts with CYCLOIDEA 2 (CYC2) TF and is involved in the regulation of reproductive organ (floral organs and pistils including style, ovary and stigma) development (Yang et al. 2019).

In future, it would therefore not be surprising to discover that WUS contributes to additional plant signaling pathways involved in other aspects of reproductive organ growth and development, and further research will undoubtedly provide novel insights for a better understanding of meristem biology in plant growth and development.

Relationship between WUS expression and shoot regeneration competence in vitro

WUS expression was shown to be essential to promote stem cell niche during shoot regeneration from cell and tissue culture studies (Meng et al. 2017; Zhang et al. 2017b). WUS is de novo activated and WUS+ expressing cells mark the shoot progenitor region during shoot regeneration in vitro (Zhang et al. 2017b).

A recent report indicated that the miR156-SPL (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE) pathway directly or indirectly represses WUS expression to regulate the SAM size (Fouracre and Poethig 2019), confirming that WUS is specifically required for stem cell identity to regulate shoot meristem identity. Noteworthy, in Arabidopsis, WUS-related homeobox WOX7 was also shown to regulate the lateral root development program in coupling with the sugar signaling, whereby WOX7 acts as transcriptional repressor and inhibits lateral root formation in a sugar dependent manner, even if it has no effect during later stages of lateral root development (Kong et al. 2016).

In a breakthrough report by Zhang et al. (2017b), a two-step model for de novo activation of WUS has been proposed, which illustrates the molecular mechanism for shoot regeneration in Arabidopsis. Using genetic and time-lapsed imaging analysis, this study revealed that during shoot regeneration the WUS+ cells mark the regenerating region in Arabidopsis. Furthermore, upon transfer to cytokinin-rich shoot inducing medium (SIM), the WUS locus is subjected to an epigenetic reprogramming (Zhang et al. 2017b). However, the molecular mechanism by which cytokinins-rich SIM medium governs such epigenetic reprogramming at the WUS locus remains elusive. Several studies also suggest that WUS activation also depends on epigenetic regulation (Li et al. 2011; Cao et al. 2015; Zhang et al. 2017b; Wang et al. 2017; Sun et al. 2019). WUS expression during de novo shoot regeneration is associated with DNA methylation and histone modifications, and it has been shown that the removal of repressive histone H3 lysine trimethylation (H3K27me3) regulates shoot regeneration by modulating WUS expression in Arabidopsis (Li et al. 2011), with such H3K27me3 removal being division-dependent and essential for WUS induction (Zhang et al. 2017b). Furthermore, genetic analysis also confirmed that epigenetic changes at the WUS locus direct WUS expression during initiation of axillary meristems (Wang et al. 2017).

Crosstalk between cytokinin signaling and WUS

Phytohormones are essential for the maintenance of shoot stem cell homeostasis, and a crosstalk exists between cytokinin action and WUS function in the SAM. Cytokinin signaling plays a key role to maintain both cell proliferation and shoot meristem activity (Riou-Khamlichi et al. 1999; Werner et al. 2003; Gordon et al. 2009; Kieber and Schaller 2018), by acting via Arabidopsis histidine kinase (AHK) receptors which further pass on the signal to two classes of TFs: type-B Arabidopsis response regulators (ARRs) and type-A ARRs (Muller and Sheen 2007). The type-B ARRs activate transcription of cytokinin-induced genes, whereas, type-A ARRs form a negative feedback loop to reduce cytokinin responses. It has been shown that cytokinin signaling directly activates a dynamic pattern of WUS expression (Gordon et al. 2009). A positive feedback loop between cytokinin signaling and WUS function influences shoot meristem patterning (Leibfried et al. 2005; Gordon et al. 2009; Chickarmane et al. 2012; Wang et al. 2017), but the underlying molecular mechanism(s) remain to be elucidated. Some reports suggest that type-B ARRs (ARR1, ARR2, ARR10, and ARR12) directly bind to the WUS promoter and induce WUS expression (Wang et al. 2017; Zhang et al. 2017a, 2017b; Meng et al. 2017; Zubo et al. 2017). Supporting this hypothesis, chromatin immunoprecipitation (ChIP) analyses confirmed that type-B ARRs directly bind to the WUS promoter (Meng et al. 2017; Zhang et al. 2017b). Genetic analysis revealed that the function of type-B ARRs is essential for WUS expression for stem cell niche during shoot regeneration (Zhang et al. 2017b; Meng et al. 2017). Interestingly, the expression of type-B ARRs was found 3 days prior to WUS in shoot regeneration (Zhang et al. 2017b). However, WUS expression can restore the shoot regenerative capacity in the arr1 and arr12 mutants (Zhang et al. 2017b). The type-B ARRs physically interact with the HD-ZIP III member of TFs and form a complex to activate WUS expression during shoot regeneration (Zhang et al. 2017b). In addition, ChIP-sequencing approach also elucidated WUS as a cytokinin-dependent direct target of ARR10 (Zubo et al. 2017). Cytokinin signaling also promotes de novo WUS expression to establish shoot stem cell niches during axillary meristem initiation (Wang et al. 2017). WUS overexpression was not found in the leaf axil in arr1-4 mutant thereby indicating that ARR1 is required for WUS activation. ARR1 binds directly to the WUS promoter region and regulates axillary meristem development through WUS activation (Wang et al. 2017).

Furthermore, WUS has been shown to repress members of the type-A ARRs, which form a negative feedback loop to regulate cytokinin signaling (To et al. 2004, 2007; Leibfried et al. 2005; To and Kieber 2008). Type-A ARRs transcript levels are also responsive to several other factors. Thus, auxin induced ARR7 and ARR15 in the RAM (Muller and Sheen 2008), but repressed them in the SAM (Liebfried et al. 2005). In a breakthrough report, To et al. (2007) discovered that the type-A ARRs interact with other proteins in a phosphate-dependent manner to produce a negative regulation of the cytokinin signaling pathway.

Most recently, Kubalová et al. (2019) showed with Arabidopsis that inducing mutations in the biosynthetic pathway of tetrapyrrole uncouples the nuclear expression of WUS from de novo shoot development. Thus, such mutants exhibit quite contrasted responses to exogenous cytokinins, with highest WUS expression coupled with the lowest shoot regeneration competence during de novo organogenesis, demonstrating that the positive tight correlation between WUS expression and SAM activity is, at least partly, regulated by tetrapyrrole intermediates.

Taken together, these results suggest that a circular interaction between cytokinin signaling and regulatory genes provides a better understanding of the regulatory feedback loop networks. However, the underlying mechanism(s) by which type-A ARRs negatively regulate cytokinin signaling is yet to be clarified.

WUS is crucial for embryogenesis

Somatic embryogenesis (SE), the in vitro transition from vegetative to embryogenic phase in plants, was first reported 60 years ago by Reinert (1958) and Steward et al. (1958) and, since then, has gained momentum. In this respect, over the last two decades, many studies have shown that several candidate TFs are differentially expressed during conversion of somatic cells into embryogenic cells (Schrader et al. 1997; Schmidt et al. 1997; Boutlier et al. 2002; Zhai et al. 2016; Kumar and Van Staden 2019), most of them not being directly involved in the vegetative to embryogenic transition, but upregulated during late embryogenesis. The only exception is SERK gene, but its function remains elusive.

WUS TF gene is reported to play a key role in plant embryogenesis. It has been found that, during SE, WUS gene is positively upregulated in several plant species, such as A. thaliana (Zuo et al. 2002), C. canephora (Arroyo-Herrera et al. 2008), G. hirsutum (Bouchabké-Coussa et al. 2013; Zheng et al. 2014) and M. truncatula (Chen et al. 2009; Orlowska and Kepczynska 2018; Tvorogova et al. 2015; 2019). Table 1 lists WUS-related genes expressed during SE in Arabidopsis, while Table 2 shows their expression in other plant species.

Table 1 WUS and WUS-related homeobox (WOX) transcription factors reported for Arabidopsis thaliana and their biological functions in plant growth regulation
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Table 2 WUS and WUS-related homeobox (WOX) transcription factors gene reported from other plant species and their biological functions in plant growth regulation
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In A. thaliana, ectopic expression of WUS was shown to be involved in vegetative-to-embryogenic transition in all tissues (leaf petiole, leaves, stem and root), without adding exogenous growth hormones (Zuo et al. 2002). Arroyo-Herrera et al. (2008) found that overexpression of WUS in C. canephora increased the somatic embryo production up to 400%, and significantly enhanced the SE in a heterologous system, but exogenous growth regulators were required for the induction of SE. In G. Hirsutum (cotton), A. thaliana WUS (AtWUS) significantly increased embryogenic callus formation (47.75%) when ectopically expressed (Zheng et al. 2014), and also positively upregulated LEC1/2 and FUS3 in such cotton embryogenic callus. Similarly, Bouchabké-Coussa et al. (2013) also revealed that overexpression of WUS gene significantly promoted (× 3) embryogenic competence and triggered in vitro regeneration capacity in cotton when WUS was expressed ectopically. However, these authors also observed that overexpression of WUS resulted in the formation of abnormal embryo-like structures and that leaf-like structures developed on the embryos (Bouchabké-Coussa et al. 2013).

WOX gene family members are expressed in the zygote and regulate cell and tissue proliferation during early embryo patterning (Haecker et al. 2004; Wu et al. 2007; Breuninger et al. 2008; Ueda et al. 2011; Zhang et al. 2017c). WOX8/9 expression is activated in the zygote and becomes restricted to the basal lineage, whereas WOX2 expression is restricted to the apical lineage where it regulates gene expression programs (Wu et al. 2007; Breuninger et al. 2008; Ueda et al. 2011). However, it is poorly understood if both genes, WOX8/9 and WOX2, are transcribed in the zygote or whether their mRNA is inherited from the egg cell. Similarly, WOX8/9 and WOX2 genes have been recognized as being expressed during early embryo development in conifers (Palovaara et al. 2010; Hedman et al. 2013; Zhu et al. 2014). Zhou et al. (2018) revealed a novel function of WOXs in regulating embryo patterning in tobacco, and confirmed by expression pattern analysis that WOX2 and WOX9 are crucial for early embryo patterning. In a very recent study with M. truncatula, Tvorogova et al. (2019) showed that the WOX9 homolog, MtWOX9-1, participates in SE and its overexpression stimulates embryogenesis capacity by changing the expression levels of various SE-associated genes.

Taken together, these studies suggest that WUS and WOX family members have a significant impact on plant biology in improving the SE capacity in plant cells. In spite of these groundbreaking discoveries in elucidating WUS expression during embryo patterning, the regulatory signaling pathways responsible for its expression and regulation remain unclear. Further studies and refinements will improve the understanding of the molecular mechanisms by which WUS expression regulates embryo patterning.

WUS-mediated transformation

Plant genetic transformation is commonly mediated by Agrobacterium, whereas microprojectile bombardment and protoplast technology (applying electroporation or chemicals such as PEG to introduce plasmids carrying the transgene) have enabled new insights in the area of plant biology, allowing direct gene transfer. For a number of species, conventional plant genetic transformation is often hampered by a low efficiency, it is time consuming, and several technical bottlenecks exist for the recovery of transgenic plants (Newell 2000; Altpeter et al. 2016; Mookan et al. 2017). However, a few recent reports have provided promising solutions in the area of plant transformation where morphogenic regulators improved the transformation efficiency in several plants (Heidmann et al. 2011; Lowe et al. 2016, 2018; Mookan et al. 2017; Jones et al. 2019).

The overexpression of WUS gene has been implicated in several model and crop species to stimulate transformation efficiency (Lowe et al. 2016; 2018; Mookan et al. 2017; Jones et al. 2019). Thus, in experiments of co-transformation of BBM with WUS2, Lowe et al. (2016) provided evidence that maize TF WUS2 stimulates high transformation frequency in several genotypes. Overexpression of maize WUS2 gene improved transformation efficiency in several monocots including Oryza sativa (ssp. indica), Saccharum officinarum calli, and Sorghum bicolor somatic embryos (immature). WUS gene was transformed directly into immature embryos or leaf segments in five different pioneer inbred lines whereby pleiotropic effects such as thickened roots, wrinkled leaves, poor functioning and sterility were observed in transgenic plants, and removal of WUS2 and BBM gene was essential to achieve normal transformants. For inbred line PHN46, a modest additional increase (4%) was found in callus transformation frequency after addition of WUS2. However, in inbred PH581, combination of WUS2 and BBM elicited an increase from 0.4% to 25.3% in callus transformation. Interestingly, inbred PHP38 showed the highest transformation frequency (51.7%) when WUS2 and BBM were used together. In line with these results, Mookan et al. (2017) found that co-expression of WUS2 and BBM TF genes stimulated efficient transformation in sorghum (P898012 genotype) and a recalcitrant maize inbred line (B73) without use of a selectable marker gene. The PHP78891 expression cassette comprised CRE:WUS2:BBM with lox P sites. Transient expression of GFP was shown in shoots, early and late embryos, pollen and vegetative organs, by introduction of Agrobacterium-mediated transgenic PHP78891 vector. Furthermore, stable transgene integration and expression in regenerated sorghum P898012 and maize B73 were confirmed by PCR and southern blotting. An enhanced transformation frequency for P898012 genotypes (6.2%) and B73 (0 to 15%) was found without the use of any selectable marker gene (Mookan et al. 2017). This selectable marker independent transformation approach may contribute to facilitate gene editing functions and overcome transformation barriers in recalcitrant genotypes. More recently, Lowe et al. (2018) showed that co-expression of WUS2 and BBM showed transformation frequencies ranging from 9.1% to 62.5% in large numbers of immature embryos in PHR03, PHH5G and PH1V69 pioneer inbred maize lines.

These findings confirmed that plant transformation technology is moving towards a new era and that morphogenic regulators may overcome many transformation obstacles. In future, the role of WUS gene can therefore be used to enhance transformation frequency and would improve the information for unknown signaling pathways stimulating transformation. In this context, new discoveries and further refinements in this area, such as the use of TF genes like BBM and WUS are likely to improve the transformation efficiency of other recalcitrant species, including monocots.

WUS functions in crops

The blooming studies of WUS in Arabidopsis and several other plant species have significantly helped in elucidating WUS functions in major crops, and offered novel insights into improvements for crop agricultural practice (Table 3). In addition to the above mentioned WUS functions on transformation efficiency in rice, maize, sugarcane and sorghum, in other species WUS is implicated in stem cell formation, somatic embryo development and floral initiation process (Chen et al. 2009; Wong et al. 2011; Zhou et al. 2018; Kyo et al. 2018; Orlowska and Kepczynska 2018).

Table 3 WUS functions in crops
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In situ hybridization analysis suggested that MtWOX5 (WUSCHEL-related homeobox gene WOX5) is expressed in auxin-induced root primordia and meristems and is involved in pluripotent stem cell formation in M. truncatula, and RNAi analysis confirmed that overexpression of MtWUS is crucial for somatic embryo production (Chen et al. 2009). MtWUS expression was induced within 2 days and further peaked after 1 week in the presence of auxin and cytokinin. However, auxin alone did not induce expression, the enhanced MtWUS expression was cytokinin-dependent and this result is consistent with WUS and cytokinin relationship in WUS-regulation of the Arabidopsis meristem (Leibfried et al. 2005), as discussed above. Another study with the model legume M. truncatula suggested an involvement of MtWUS and MtWOX5 during the initiation phase of SE, when they were key markers for cell dedifferentiation in leaf explants both in M9 (non-embryogenic) and M9-10a (embryogenic) lines (Orlowska and Kepczynska 2018). Thus, MtWUS expression on both lines was found to be maximum at day 2 (120-fold higher), but it decreased rapidly after 7 days. Meanwhile, an extreme upregulation of WOX5 expression occurred between day 3 and day 14 and it remained unchanged thereafter (Orlowska and Kepczynska 2018). Similar to M. truncatula, in tobacco (N. tabacum) WOX genes are involved during early embryogenesis (Zhou et al. 2018), most of them exhibiting a cell type-specific and stage-specific expression pattern during embryogenesis. RT-qPCR (Quantitative real time reverse transcription PCR) analyses revealed that WOXs genes (WOX2 and WOX9) are crucial for early embryo patterning (Zhou et al. 2018). A recent study also showed that coexpression of WOXs (WOX2, WOX8 and WOX9) promotes remarkable regeneration from freely suspended cells and leaf segments of tobacco (Kyo et al. 2018).

In soybean (Glycine max), WUS spatial expression in the incipient floral primordia elucidated WUS function in the floral initiation process (Wong et al. 2011). RT-PCR analysis revealed that GmWUS (soybean ortholog of WUS) is expressed in the SAM and floral meristem, while in situ hybridization showed that GmWUS accumulates in incipient floral primordia. These observations are largely consistent with those reported earlier for the initiation of floral primordia in Arabidopsis (Wagner 2009). Interestingly, ectopic expression of GmWUS is sufficient to produce adventitious shoot formation on the petiole of a rosette leaf, whereas disruption in floral organ formation includes missing petals, defective floral buds and normal stamens and carpels (Wong et al. 2011). These results are also consistent with those reported for Arabidopsis WUS (Xu et al. 2005; Ikeda et al. 2009; Lohman et al. 2001).

It appears that WUS mediates the stress response and regulates early flowering in rice (Minh-Thu et al. 2018). RT-PCR analysis confirmed that OsWOX13, a homeodomain TF, was moderately upregulated under drought stress in leaf and root of rice. Overexpression of OsWOX13 triggered floral development resulting in 7–10 days earlier flowering in rice (Minh-Thu et al. 2018). The OsWOX4 member of WOX gene family regulates cellular activity in leaf development including tissue differentiation of both vascular development and midrib formation, as transcriptome profiling revealed that OsWOX4 regulates the expression of several genes in leaf primordia and promotes cell proliferation, leading to leaf development (Yasui et al. 2018). The WOX3 gene, LEAF LATERAL SYMMETRY1 (LSY1), is involved in lateral organ development in rice by regulating adaxial-abaxial patterning at the edge of leaf primordia, and LSY1 also regulates trichome initiation and function in the inflorescence by maintaining adaxial-abaxial identity in the stamens (Honda et al. 2018). Most recently, Hao et al. (2019) showed that the overexpression of GmWOX18 significantly increased (more than 150-fold) adventitious shoot bud regeneration capacity in soybean under different abiotic stresses.

The summation of these discoveries on WUS and WUS-related homeobox genes provides novel mechanistic insights into the development of several crop species. However, further studies on the molecular regulation mechanisms underlying the functions of WUS and WOX genes will also facilitate an understanding of the diversification of WUS and WOX genes in different plant species, including monocots and eudicots for a better improvement of their sustainable agricultural performance.

Concluding remarks and perspectives

Our understanding of the molecular mechanisms which govern meristem regulatory networks and other plant signaling pathways is constantly increasing. A number of groundbreaking researches in Arabidopsis and various crop species have significantly increased our understanding of meristem biology. WUS expression has significant potential on plant biology research and other biotechnological applications. Several discoveries bridge the gap between WUS expression and plant signaling pathway by identifying different WUS and WUS-related homeobox genes during the formation of shoot (apical and axillary) meristems, vegetative-to-embryo transition, genetic transformation, and other aspects of plant growth and development.

The studies discussed above suggest that the WUS gene is required for meristem identity by recruiting transcriptional corepressors that induces differentiation and maintenance of stem cells. WUS was shown to be involved in vegetative-to-embryogenic transition without adding any exogenous growth hormones, when expressed ectopically. We also note that the WOX family of TFs comprises multiple members which are expressed in the zygote and involved in diverse signaling pathways during early embryo patterning. In addition, overexpression of WUS stimulates high transformation frequency in several genotypes, even if the rather limited information regarding transformation efficiency is due to the unknown signaling pathway which still remains unclear.

In addition to the above mentioned WUS functions, in crop species WUS and WOX genes are implicated in leaf development including tissue differentiation of both vascular development and midrib formation, lateral organ development, trichome initiation and function in the inflorescence by maintaining adaxial-abaxial identity in the stamens.

Further experiments should shed some light on how these regulatory members co-ordinate and control meristem biology and several aspects of plant dynamics. To decode these regulatory networks, a single-molecule imaging technology will be required to understand the diverse functions of individual WOXs in different signaling pathways. Together, structural studies of different WOXs may open new avenues for better understanding their signaling specificity and developmental plasticity.

During the past decade, new signaling pathways of WUS TF gene regulating diverse biotechnological functions of plant growth and development have been discovered. However, we are far from understanding the molecular mechanism(s) and complex network(s) of WUS TF signal(s) that still need to be deciphered, and how these TFs integrate cell-to-cell communication and regulate cell behavior in several plant growth responses. For future research, several tools have emerged, such as single-molecule imaging technology, that may help reveal important details of signaling pathways. In addition, further research will provide novel insights for a better understanding of meristem dynamics in plant growth regulation, which should in turn improve the modern biotechnological approaches for agriculture and crop productivity.

Recent discoveries have explored the multiple roles of WUS in diverse aspects of plant growth and development. However, a few outstanding questions still need to be clarified: (1) WUSCHEL-related homeobox (WOX) family contains multiple members which are involved in the diverse signaling pathways related to meristem regulatory network, but how these pathways are regulated remains unclear. (2) Individual WOX members regulate differentially to different signaling pathways. How is the specificity of these different WOXs members obtained? (3) Apart from known processes, what additional physiological and biological processes are regulated by WUS? (4) The underlying molecular mechanism by which WUS function and cytokinin signaling make a positive feedback loop and regulates shoot meristem patterning is still unclear.