The WUSCHELa (PtoWUSa) is Involved in Developmental Plasticity of Adventitious Root in Poplar

WUSCHEL-RELATED HOMEOBOX (WOX) transcription factors play critical roles in cell fate determination during plant development. As the founding member of the WOX family, WUSCHEL (WUS) is characterized for its role in maintaining stem cell in meristem. In this study, we investigated the function of Populus tomentosa WUSCHELa (PtoWUSa) in adventitious roots (ARs) in poplar. Expression profile analysis showed that PtoWUSa was not only expressed in shoot apical meristem and stem, but also expressed in ARs. Ectopic expression of PtoWUSa in Arabidopsis resulted in shortened primary root, as well as agravitropism and multiple branches. Overexpression of PtoWUSa in poplar increased the number of ARs but decreased their length. Moreover, the AR tip and lateral root tip became larger and swollen. In addition, the expression of auxin transporter genes PIN-FORMED were downregulated in ARs of transgenic plant. Taken together, these results suggest that PtoWUSa could be involved in AR development in poplar through regulating the polar auxin transport in ARs.


Introduction
Cutting is a main method of vegetative propagation for woody plants. An essential step of cutting depends on the formation and development of adventitious roots (ARs). Therefore, dissecting the molecular mechanisms of the development of ARs and exploring the function of genes regulating adventitious rooting will be beneficial for improving propagation efficiency. ARs are always generated from the stem-, hypocotyl-or leaf-derived cells. The development of ARs is regulated by multiple endogenous and environmental factors. Among them, phytohormone auxin plays a key role [1]. Auxin is mainly synthesized in young aerial parts and flows down to the root tip columella in which an auxin maximum is formed, and then transports to other parts of the root through root epidermal cells [2]. During this process, the formed auxin concentration gradients regulate the root growth and plasticity. The auxin transport from cell-to-cell is mediated by auxin transport carriers, such as AUX1-like influx carrier, PIN-FORMED efflux carrier (PIN) and P-glycoprotein auxin carrier (PGP) [3]. The PIN is characterized by its polar plasma membrane localization that regulates the direction of auxin flow [4]. PGPs can improve the stability of PINs in plasma membrane and enhance cellular auxin transport activity [5]. PIN, PGP efflux transporters, and AUX1 influx transporters determine the directionality of intercellular auxin flow [6]. One of the prominent growth responses regulated by auxin polar transport is root tropism, such as the mutants of PIN2, PIN3, PIN5, PIN7, and AUX1 with root gravitropism [7][8][9][10][11].
The WUSCHEL-related homeobox (WOX) gene family is a plant-specific transcription factor and belongs to the homeodomain superfamily [12]. The function of WOX genes in Arabidopsis thaliana have been well characterized and show that WOXs play critical roles in stem cell maintenance in shoot and root apical meristems (SAM and RAM), root development, et al. [13][14][15]. AtWOX1/AtWOX3 regulates lateral axis-dependent development of lateral organs [16]. AtWOX2, AtWOX8 and AtWOX9 are required for apical-basal axis formation of pre-embryo, and AtWOX9 also plays a role in maintaining the cell division in early embryogenesis [17,18]. AtWOX4 is expressed in vascular and regulates the vascular stem cell proliferation, and it acts redundantly with AtWOX14 in the maintenance of vascular meristem organization during secondary growth [19,20]. AtWOX5 is specifically expressed in the cells of quiescent center and functions in organizing stem cells in RAM [21,22]. AtWOX7 regulates the lateral root (LR) development [23]. AtWOX11/12 are involved in AR de novo organogenesis by activating the expression of AtWOX5 and AtWOX7 [24,25].
As the founding member of WOX family, WUSCHEL (WUS) is originally identified in Arabidopsis [26]. AtWUS serves as a central regulator in the maintenance of stem cell identity in the central zone of SAM and floral meristems [26,27]. AtWUS expression is restricted in organizing center of SAM, and AtWUS protein can move to the stem cell region to promote the expression of CLAVATA 3 (CLV3), then the activated CLV3 signaling pathway inhibits the expression of WUS, resulting in maintaining stem cell homeostasis in SAM [28][29][30]. At the reproductive phase, the WUS-AGAMOUS feedback loop controls the flower meristem (FM) identity [31]. In atwus mutant, plants develop in stop-and-go mode, and defective SAM and FM are initiated repetitively, leading to many disorganized bunches of leaves along the plant [26]. In Oryza sativa, OsWUS is expressed in young leaf primordia, axillary meristems (AMs) and transient expressed in rice SAM [32][33][34]. OsWUS is required for initiation of the AMs, but not for maintenance of the SAM [34]. The mutants of OsWUS, tab1-1 [24], moc3-1 [33], and srt [35] exhibit various degree defects in tillering and flower development. Overexpression of cotton (Gossypium hirsutum) GhWUS can induce somatic embryo and shoot formation [36]. Despite the function of WUS in developmental processes, which has been studied in herbaceous plant, little is known about its function in woody plants.
As a model woody species, Populus is characteristic with rapid growth and high production of plant biomass. In our previous study, 18 WOX genes were identified in Populus tomentosa, and 2 P. tomentosa WOXs (PtoWUSa and PtoWUSb) were identified as the orthologous genes of Arabidopsis AtWUS [37]. In this study, we characterized the function of PtoWUSa in AR development. Expression profile analysis showed that PtoWUSa was not only expressed in SAM and stem, but also expressed in ARs. Transgenic results displayed that overexpression of PtoWUSa affected the root morphogenesis by down-regulation of PIN expressions.

Plant Materials and Growth Conditions
A. thaliana (ecotype Columbia-0) was grown at 20-22 • C under 16 h/8 h light/dark photoperiod in 1/2 Murashige-Skoog (MS) medium or soil. Hybrid poplar (P. alba × P. glandulosa) 84K was performed tissue culture to obtain poplar clonal plants for transgenic acceptors and was grown at 23-25 • C under 16 h/8 h light/dark photoperiod.

RNA Extraction and qRT-PCR Assays
Total RNAs were extracted from plant samples using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The quality and concentration of RNAs were detected by agarose gel electrophoresis and NanoDrop 8000 (Thermo, Wilmington, USA). The first stand cDNA was acquired using SuperScript III first-strand synthesis system (Life technologies, Carlsbadn, USA) with 1 µg mRNA. Quantitative real-time PCR (qRT-PCR) was performed on the Roche LightCycler 480 (Roche Applied Science, Penzberg, Germany) following manufacturer's instructions. Actin7 and PtActin were chose as reference genes. The 2 −∆∆CT method was used to calculate the relative expression level [38]. Three biological replicates and four technical replicates of each sample were performed for qRT-PCR analysis. The primers used in this study were listed in Table S1.

Plasmid Construction and Transformation
The coding sequence and the promoter of PtoWUSa were amplified and cloned into pDNOR222.1 for sequencing. The right coding sequence was sub-cloned respectively into pMDC32 and pMDC7, represented as p35S::PtoWUSa and pXVE::PtoWUSa. The right promoter sequence was conducted into pMDC164, represented as pPtoWUSa::GUS. The resulting constructs were then transformed into Agrobacterium GV3101 by electroporation. Agrobacterium carrying pPtoWUSa::GUS, p35S::PtoWUSa, or pXVE::PtoWUSa were used for poplar transformation. The RNAs were extracted from roots of two-week-old p35S::PtoWUSa transgenic A. thaliana plants and three-week-old p35S::PtoWUSa transgenic poplar plants, and the transcription levels of PtoWUSa were detected using qRT-PCR. Two independent transgenic A. thaliana lines (#31 and #43) and two independent transgenic poplar lines (#16 and #18) with high PtoWUSa abundance were used for further study. The pPtoWUSa::GUS was used for GUS staining according to the previous studies [39].

Estradiol Treatments
The 17 β-estradiol was dissolved in dimethyl sulfoxide (DMSO) as 10 mM stock solution. The apicals (2-3 cm) from 84 K and two independent pXVE::PtoWUSa transgenic lines (#26 and #27) with two leaves were induced by adding 17 β-estradiol into the nutrient solution, while the untreated condition was corresponded to plants treated with DMSO. Phenotypic observation was carried out on 20-day-old poplars. The concentration of 17 β-estradiol was determined based on treatment in poplar [40].

Statistical Analysis
The statistical analysis of all the experimental data was performed with SPSS 16.0 software (SPSS Inc, Chicago, USA). Significant differences were detected using t test and the differences were considered significant if p < 0.05.

Phylogenetic Analysis and Protein Sequence Comparisons
Two P. tomentosa WOXs (PtoWUSa and PtoWUSb) were identified as the orthologous genes of AtWUS in our previous study, but only PtoWUSa was expressed in AR [37]. Therefore, PtoWUSa was chosen for function investigation in AR development in the present study. PtoWUSa contained a helix-loop-helix-turn domain in the N-terminal which was known as the homeodomain (HD), an EAR-like domain in the C-terminal, and a conserved WUS-box domain (TLxLFP) between them ( Figure 1). The result of phylogenetic relationships between PtoWUSa and AtWOXs showed that PtoWUSa belongs to the WUS/modern clade and had a close genetic relationship with AtWUS ( Figure S1). Compared with WUS protein sequences from different species, PtoWUSa shared 29.83-98.86% identity with WUS sequences from other species (Figure 1). PtoWUSa and PtrWUSa shared 98.86% identity and had only three residues difference over the entire amino acid sequence. These results indicate that WUS had conserved domain, although the sequences were variable in different plant species.

Expression Pattern of PtoWUSa
The GUS reporter of PtoWUSa was conducted to confirm the expression pattern of PtoWUSa. GUS staining, showing that the GUS signals were detected in the SAM ( Figure 2A) and stem ( Figure 2B) and roots ( Figure 2C-F). The roots from the poplar stem were named ARs, and the roots located in the ARs were named LRs. In roots, the GUS signal was specially detected in the AR tip (ART) and the LR tip (LRT) (Figure 2C-F). These results indicated that the PtoWUSa accumulated in LRT and ART in the roots, suggesting that PtoWUSa might play roles in root development.

Ectopic Expression PtoWUSa Affected Root Development in Arabidopsis
As the herb model plant, Arabidopsis has a rapid lifecycle and easy transformation system. Therefore, we firstly investigated the function of PtoWUSa by ectopic expression of PtoWUSa in Arabidopsis. Two independent transgenic lines (#31 and #43) with high abundance of PtoWUSa ( Figure 3G) were chosen for functional study. After cultured on 1/2 MS medium for two weeks, the length of primary roots of PtoWUSa-overexpressing Arabidopsis seedlings was severely inhibited and was only 50% of WT seedlings ( Figure 3A-C,H). Interestingly, the roots of transgenic plants appeared to curve and showed agravitropism ( Figure 3A-C), and their root tip was enlarged and swollen ( Figure 3D-F). After three weeks of growth, the abnormal root morphology of transgenic plants was intensified, such as the decreased primary root length, agravitropism, and curved root ( Figure 4). The root hair and LRs were located closer to the root tip in transgenic plants, being different with WT plants ( Figure 4F,H). It is noteworthy that the LR length of transgenic plants was increased compared with WT plants when the transgenic LRs were stretched out ( Figure 4B).  Moreover, the PtoWUSa-overexpressing Arabidopsis plants exhibited more branches than WT plants ( Figure 5A-D), and the stem number of transgenic plants reached to four to six ( Figure 5F), but the height of primary stem of transgenic plants was reduced to 1/3-1/2 of WT plants after growing on soil for four weeks ( Figure 5E).

Overexpression of PtoWUSa also Alters the Morphology of ARs in Poplar
In order to investigate whether PtoWUSa performed similar functions in poplar, we generated transgenic poplar plants overexpressing PtoWUSa. Two independent transgenic lines (#16 and #18) with high abundance of PtoWUSa were used for further study. After cultured for three weeks, the number of ARs increased to 6 to 8 in transgenic seedlings, which was about twice in control (Ctrl) ( Figure 6A-C). Additionally, the AR length of PtoWUSa-overexpressing poplar plants (~2 cm) was significantly less than that of Ctrl plants (~6 cm) ( Figure 6A-C). Moreover, the morphology of ARTs and LRTs in Ctrl plants was always sharp and small, whereas ARTs and LRTs became enlarged and swollen in transgenic plants. The location of LRs was closer to the ARTs in transgenic plants than in Ctrl plants (Figure 7).  To further confirm the functions of the PtoWUSa, we constructed an inducible expression vector pXVE::PtoWUSa and transformed it into poplar. After being induced under 10 µM 17 β-estradiol for 15 days, the two transgenic lines (#26 and #27) with high expression of PtoWUSa were chose for subsequence study ( Figure 8D). There was no different phenotype between the Ctrl and transgenic plants without 17 β-estradiol treatment ( Figure 8A-C). After treatment by 17 β-estradiol, the AR primordia was observed, AR elongation was inhibited and the ARTs became enlarged and swollen in the pXVE::PtoWUSa transgenic plants ( Figure 8E-I). All these results indicated that PtoWUSa was involved in AR development in poplar.

Expression Analysis of Auxin-Related Genes in PtoWUSa-Overexpressing Transgenic Poplar
The extensive researches have been shown that auxin acts as an important regulator in root development. The root morphology of transgenic plants overexpressing PtoWUSa was similar to the pin or aux mutants. This observation impelled us to clarify whether this phenotype in the present study was caused through altered distribution of auxin in transgenic plants. Thus, we examined the expression of auxin transport genes including AUX and PIN in the ARs of three-week-old PtoWUSa-overexpressing plants and Ctrl plants. The expression levels of eleven PINs and five AUXs were successfully detected. Among PIN genes, the expression levels of five PIN genes (PIN1c, PIN2, PIN3b, PIN5a, and PIN6a) for auxin polar transport were reduced, while other genes were slightly up-regulated ( Figure 9). The expression levels of AUXs were no obvious change (Figure 9). These results suggested that overexpression of PtoWUSa might affect polar transport in the ARs by regulating the expression of PINs.

Discussion
WUS is the founding member of the WOX family, which is the plant-specific transcription factor. It has been well documented that WUS is expressed in SAM and FM, and its main function is to maintain stem cell balance and to keep stem cells balance between division and differentiation in herbage plant. However, research on the homologues of WUS in woody plant is lacking and its function is unclear. In this study, the function of poplar PtoWUSa was characterized. The result of sequence analysis showed that PtoWUSa was a member of the WUS clade containing the conserved domains of HD, WUS-box, and EAR-like domain. Expression patterns exhibited that PtoWUSa was not only expressed in SAM, but was also expressed in root and stem, which was different from expression patterns of other WUS that have been reported. It has been confirmed that WUS also plays role in its expressed zone, such as flower and shoot meristem in Arabidopsis [26,27]. Therefore, its expression patterns indicated that PtoWUSa might participate in root and stem development.
In our study, multiple stems were present in PtoWUSa-overexpressing plants, implying that PtoWUSa affected the meristem growth. Moreover, the root development was disordered in Arabidopsis and poplar, indicating that PtoWUSa was involved in AR development. It is reported that WUS play key role in shoot regeneration. The atwus mutant is defective in shoot meristem development, and overexpression of AtWUS can promote the transition from vegetative stage to embryonic stage, and GhWUS is also involved in the formation of the embryogenic callus in cotton [36]. However, being different in AtWUS and GhWUS transgenic plants [36,41,42], embryo-like structures were not detected in the root tip of PtoWUSa-overexpressing Arabidopsis and poplar plants. These results implied that PtoWUSa might have a weaker function in somatic embryogenic than AtWUS and GhWUS.
WOX transcription factors have been found to be involved in AR development. In Arabidopsis, AtWOX7 from WUS-clade is involved in LR development and it is activated by AtWOX11/12 in the process of AR de novo organogenesis [25,37]. However, no AtWOX7 homolog gene was identified in poplar. Based on the expression pattern and evolutionary relationship, we speculated that PtoWUSa, PtoWOX5a or other PtoWOXs might take place of the roles of WOX7 in poplar AR development. In poplar, a member of WUS clade, PtoWOX5a also expressed in the ARs, and an overexpression of PtoWOX5a affected the morphology of ARs and LRs [39]. WOX5 can rescue the deficiencies of wus mutants in Arabidopsis, indicating that WUS and WOX5 has similar function [43]. Furthermore, the similar phenotype of ARs in PtoWUSaand PtoWOX5a-overexpressing plants indicated that PtoWUSa and PtoWOX5a also had similar function in AR development in poplar. The direct genetic evidences including CRISR-cas9 or RNAi need to further reveal its function in AR development in future.
In A. thaliana, WUS has been demonstrated to be involved in meristem growth and maintenance via acting its down-stream genes, such as CLV3 and type-A Arabidopsis Response Regulator 5/7/15 (ARR5/7/15). In SAM, WUS can directly activate CLV3 and in turn the CLV3 signal represses WUS expression. This WUS-CLV feedback loop controls SAM maintenance [28,29]. WUS is linked to cytokinin signaling through direct transcriptional repression of ARR5/7/15, which are negative regulators of cytokinin signaling and are necessary for proper meristem function [44]. In our study, we also found that overexpression of PtoWUSa repressed the expression of five PIN genes (PIN1c, PIN2, PIN3b, PIN5a and PIN6a) in ARs in poplar. PIN auxin efflux carriers and AUX1-like auxin influx carriers are two classes of proteins for polar auxin transport [10]. The PIN function has been confirmed to take part in polar auxin transport, which is important for root morphogenesis [2,45]. OsPIN1 is expressed in root primordial, and suppressing the expression of OsPIN1 significantly inhibits ARs development through affecting auxin transport [8,46]. AtPIN2 plays an important role in controlling gravitropism by regulating the redistribution of auxin from the stele towards the elongation zone of roots. In pin2 mutant, the root showed agravitropism and reduced elongation [7]. In our previous study, it has been confirmed that PtPIN1, PtPIN2, and PtPIN5a are specifically expressed in ARs, implying that they are involved in AR development by regulating the polar auxin transport in ARs [47]. In PtoWUSa-overexpressing plants, the expression levels of five PtPINs (PIN1c, PIN2, PIN3b, PIN5a and PIN6a) were down-regulated, and this might influence polar auxin concentration and result in agravitropism of transgenic plant (Figures 4 and 5). In the pin2 and pin5 mutants, the root elongation was inhibited [7,48]. The reduced expression of PtPIN5a and PtPIN2 might be the reason why the AR elongation was inhibited in PtoWUSa-overexpressing plant in our study ( Figure 6). No obvious change of expression levels of AUX1-like suggested that PtoWUSa was mainly regulated the efflux transport of auxin, rather than auxin influx carrier. These studies indicate that PtoWUSa may have superimposed coordination of reprogramming towards developmental plasticity, and it processes multiple regulatory mechanisms and biological functions. Further investigations need to be carried out to reveal its complex regulatory network in future.

Conclusions
In this study, we explored the function of PtoWUSa and found it is involved in AR development by down-regulating the expression of auxin efflux carrier in PtoWUSa-overexpressing plants. Constitutive expression of PtoWUSa caused the defect of root development, including agravitropism, and enlarged and swollen root tip in transgenic Arabidopsis and poplar plants. Our results provide some new information about function of WOX in poplar, and this study will help us gain a better understanding the AR development in woody plant.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/11/2/176/s1. Figure S1. Phylogenetic analysis of PtoWUSa and AtWOXs. The 18 proteins were clustered into three distinct groups: the modern/WUS clade, an ancient clade, and an intermediate clade. Table S1. The primers sequences used in this study.

Conflicts of Interest:
The authors declare no conflict of interest.