WOX14 promotes bioactive gibberellin synthesis and vascular cell differentiation in Arabidopsis

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WOX14 promotes bioactive gibberellin synthesis and

vascular cell differentiation in Arabidopsis

Erwan Denis, Nadia Kbiri, Viviane Mary, Gaëlle Claisse, Natalia Conde E

Silva, Martin Kreis, Yves Deveaux

To cite this version:

Erwan Denis, Nadia Kbiri, Viviane Mary, Gaëlle Claisse, Natalia Conde E Silva, et al.. WOX14

promotes bioactive gibberellin synthesis and vascular cell differentiation in Arabidopsis. Plant Journal,

Wiley, 2017, 90 (3), pp.560-572. �10.1111/tpj.13513�. �hal-01569515�

WOX14 promotes bioactive gibberellin synthesis and

vascular cell differentiation in Arabidopsis

Erwan Denis1_{, Nadia Kbiri}1_{, Viviane Mary}1_{, Ga€elle Claisse}1_{, Natalia Conde e Silva}1,2_{, Martin Kreis}1_{and Yves Deveaux}1,2,_* 1_{Saclay Plant Science, Institut de Biologie des Plantes, Univ. Paris-Sud, CNRS, Orsay 91405, France, and}

2_{GQE– Le Moulon, INRA, Univ. Paris-Sud, CNRS, AgroParisTech, Universite Paris-Saclay, Gif-sur-Yvette 91190, France}

Received 16 July 2015; revised 6 February 2017; accepted 8 February 2017; published online 20 February 2017. *For correspondence: (e-mail yves.deveaux@u-psud.fr).

SUMMARY

Procambial and cambial stem cells provide the initial cells that allow the formation of vascular tissues. WOX4 and WOX14 have been shown to act redundantly to promote procambial cell proliferation and differ-entiation. Gibberellins (GAs), which have an important role in wood formation, also stimulate cambial cell division. Here we show that the loss of WOX14 function phenocopies some traits of GA-deficient mutants that can be complemented by exogenous GA application, whereas WOX14 overexpression stimulates the expression of GA3ox anabolism genes and represses GA2ox catabolism genes, promoting the accumulation of bioactive GA. More importantly, our data clearly indicate that WOX14 but not WOX4 promotes vascular cell differentiation and lignification in inflorescence stems of Arabidopsis.

Keywords: WOX, gibberellin, cambium, cell differentiation, xylem, inflorescence stem, Arabidopsis thaliana.

INTRODUCTION

Vascular meristems are responsible for the formation of the conductive tissues that interconnect all plant organs in a continuous network. Xylem and phloem are specified from the procambium, and are organized into precisely patterned vascular bundles (VBs). Secondary growth starts with the formation of the cambial meristem that originates from the procambium and the interfascicular parenchyma cells (Ye, 2002; Sieburth and Deyholos, 2006). The underly-ing mechanisms require interplay between different signal-ing molecules and regulators, such as plant hormones and small peptides (Elo et al., 2009; Miyashima et al., 2013; Nieminen et al., 2015).

Auxin, gibberellin (GA) and cytokinin are the best-charac-terized hormonal regulators of vascular development. The specification and patterning of continuous vascular strands are driven by auxin fluxes (Iba~nes et al., 2009). Indeed, plants that overproduce auxin molecules have increased levels of vascular tissues, and local auxin application can induce the formation of new vascular strands from parenchymatic cells (Mattsson et al., 2003). Gibberellins are important regulators of wood formation, and are thought to be the mobile signal that drives the transition from primary to secondary growth (Mauriat and Moritz, 2009; Ragni et al., 2011; Dayan et al., 2012). Bioactive GA signaling relies on the proteolysis of DELLA proteins, and its steady-state level

depends on the activity of GA activation and deactivation enzymes coded by the GA3-oxidase (GA3ox) and GA2-oxi-dase (GA2ox) gene families, respectively (Yamaguchi, 2008; Daviere and Achard, 2013). It has been suggested that the main function of GA during wood formation is to regulate the early phase of xylem cell differentiation and elongation, as both DELLA proteins and bioactive GA accumulate in these cells (Israelsson et al., 2005; Tokunaga et al., 2006). Other studies, however, have indicated that GA is not only involved in cell differentiation but also in cell proliferation, as DELLAs also function in limiting cell production (Achard et al., 2009). Additionally, GA has been suggested to stimu-late cambial cell proliferation in poplar by inducing the tran-scription of the auxin transport gene PIN1 (Bj€orklund et al., 2007). There is likely to be an interplay between GA and auxin for the control of VB patterning, as auxin has also been shown to regulate GA synthesis (O’Neill et al., 2010). Last but not least, cytokinin is also important for cambial cell proliferation, and acts synergistically with auxin and GA to coordinate vascular development (Matsumoto-Kitano et al., 2008; Nieminen et al., 2008).

In Arabidopsis, the ligand/receptor pair CLAVATA3/ESR LIKE 41/PHLOEM INTERCALATED WITH XYLEM (CLE41/ PXY), also called TDIF/TDR, constitutes one of the path-ways that control cell proliferation and differentiation of

the cambium and its derivatives (Fisher and Turner, 2007; Hirakawa et al., 2008). Expression studies of both CLE41 and PXY have led to the proposal of a non-cell autono-mous model in which the peptide ligand CLE41 is pro-duced by phloem cells and secreted into the cambium. Once bound to the PXY receptor, CLE41 promotes the maintenance of a stem cell pool by enhancing cell division and suppressing cambial cell differentiation into xylem cells (Hirakawa et al., 2010a). Such regulation maintains continuous xylem production by preventing the precocious consumption of cambial cells. A recent study showing that cambial division rate and wood formation are enhanced when the CLE41/PXY expression profile is altered in hybrid aspen (Etchells et al., 2015) is consistent with this model.

Control of the balance between stem cell proliferation and differentiation has been proposed to be the conserved function of the WOX gene family (Laux et al., 1996; Haecker et al., 2004; Nardmann and Werr, 2012). The WOX family, divided into three phylogenetic clades, has several members that are expressed in vascular tissues (Deveaux et al., 2008). In Arabidopsis, WOX4 has been shown to be the integrator of the CLE41/PXY signal that controls vascu-lar cell proliferation (Hirakawa et al., 2010b). Recently, WOX14 has been suggested to act redundantly with WOX4 to control vascular cell proliferation, and both genes have been found to be regulated by the CLE41/PXY pair (Etchells et al., 2013). Whereas WOX4 is a member of the most recent clade that also includes the WUS and WOX5 genes, known to be regulators of shoot and root meristems, WOX14 belongs to the most ancestral clade (Deveaux et al., 2008). This finding further suggests that the control of meristematic cell division and differentiation by CLE-WOX genes is a conserved mechanism.

Different studies have also suggested that WOX genes might be the genetic factors that link the CLE peptide sig-naling pathway and hormonal cues: (i) WOX genes have been reported to be repressors of cytokinin receptor gene expression within plant primary meristems (Leibfried et al., 2005; Zhao et al., 2009); (ii) cambium stimulation by auxin is dependent on a functional WOX4 (Suer et al., 2011). As GAs have an important role in vascular development, it is pertinent to examine whether some of the WOX genes that are expressed in vascular tissues also interact with the GA pathway to integrate the CLE/PXY pathway.

In this study, using both mutant and overexpressing lines, we provide evidence of a link between WOX14 func-tion and the GA biosynthesis pathway. Whereas wox14 plants showed several phenotypes that suggested an alter-ation of GA biosynthesis or signaling, WOX14 overexpres-sion increased bioactive GA production and induced radial growth with strong lignification of vascular stem tissues. Moreover, we show that WOX14 but not WOX4 promotes vascular cell differentiation in the inflorescence stem of Arabidopsis.

RESULTS

The wox14 late-flowering phenotype is rescued by exogenous GA application

Initial characterization of the null wox14 mutant revealed a number of phenotypes that suggest a deficiency in GA pro-duction or signaling. During the vegetative phase, as observed in GA-deficient Populus plants (Gou et al., 2010), 10-week-old soil-grown wox14 plants showed an increased lateral root biomass under short-day conditions, mainly in the adventive root zone (Figure S1a). The wox14 plants also showed a delay in floral transition under long-day conditions, having seven additional leaves at floral transi-tion compared with the control plants (Figure 1a, b).

As GA has been suggested to be a positive regulator of Suppressor of Overexpression of Constans 1 (SOC1) and a negative regulator of Flowering Locus C (FLC) (Moon et al., 2003; Zhang et al., 2009), transcript levels of these flower-ing regulators were measured in both mutant and wild-type plants. In 6-day-old plants grown in vitro, FLC tran-script levels were two times higher than in the mutant line, whereas no significant difference was observed in 14-day-old plants (Figure 1c). SOC1 expression strongly increased between days 6 and 14 in the control plants, whereas only a weak increase was measured in the mutant line (Fig-ure 1d). Hence, the higher initial level of FLC and the weak upregulation of SOC1 are in agreement with the wox14 late-flowering phenotype, and support the hypothesis of a deficit in GA content. We therefore expected that exoge-nous GA should rescue the wox14 late-flowering pheno-type. Indeed, when exogenous GA3 was applied to 7-day-old soil-grown plants until flowering, both wox14 and con-trol plants had an average of 10 leaves at floral transition (Figure 1b). Additionally, as SOC1 integrates both the FLC and GA pathways (Moon et al., 2003), we also expected that prolonged seed stratification should overcome the high FLC levels and restore flowering time, even in the absence of exogenous GA (Chiang et al., 2009). As expected, a 6-week period of stratification at 4°C in the dark fully complemented the wox14 late-flowering pheno-type (Figure 1b).

WOX14 expression accumulates within the procambium during stem maturation and promotes xylem

differentiation

Our quantitative PCR analysis along the stem indicated that WOX14 accumulates during stem maturation (Fig-ure S1b), which is consistent with previous data (Etchells et al., 2013). It has also been previously shown that WOX14 is expressed during lateral root emergence and within the vasculature during vegetative development (Deveaux et al., 2008). We therefore monitored the spa-tiotemporal transcriptional activity of the WOX14 promoter during floral transition and bolting. At floral transition, the

WOX14 promoter was activated in tissues of the medullar zone, with its activity becoming strong and restricted to the vascular strand (Figure S1c). In stem sections made above the rosette leaves, GUS expression was detected only in the fascicular meristem, with its levels increasing during stem maturation (Figure 2a–c), confirming the puta-tive function of WOX14 in vascular stem development and maturation.

Hand sections of stems showed that in young stems the mutant line produced more VBs than the wild type (Fig-ure 3a, b). We also noticed that in sections of stems at early stages of maturation, the lignification of VBs was weaker in wox14 compared with the wild type (Figure 3c, d). This phenotype was confirmed in more mature tissues, as sections from the base of mature inflorescence stems also had on average three additional VBs (Figure S2a), and showed lower lignification (Figure 3e, f) in wox14. Tolu-idine blue-stained thin sections confirmed the reduced pro-duction of xylem fibers in wox14 mature stems (Figure 3g, h). Here as well, exogenous GA application was able to rescue the wox14 extra-VB phenotype, with both control and mutant plants having eight strongly lignified VBs under GA treatment (Figure 3i, j).

Such strong phenotypes were not reported in a previous study that used another wox14 line with a T-DNA insertion in the middle of the first intron (Etchells et al., 2013), whereas we used the pst13645-wox14 line that has an insertion within the first exon (Deveaux et al., 2008). Never-theless, complementation assays of pst13645-wox14 con-firmed the role of WOX14 in floral transition and xylem differentiation. Using the WOX14 cDNA under the control of a 1-Kb WOX14 promoter fragment, we fully rescued both the late-flowering and the extra-VB phenotypes of the mutant line (Figure S2). To further confirm that WOX14 promotes xylem differentiation and floral transition, WOX14 cDNA was constitutively expressed under the con-trol of the strong 35S promoter to generate independent WOX14 overexpressing lines (OEWOX14; Figure S3a, b). On soil, OEWOX14 plants did not show any striking pheno-type before bolting; however, they produced floral stems that had a slower elongation rate (Figure 4a) correlated with greater stiffness (Figure 4b) compared with the wild type. OEWOX14 stems were also thicker, with the stem base diameter of 7-week-old plants being on average 27% wider (Figure 4c). Stem cross-sections stained with phloroglucinol revealed a precocious and increased forma-tion of strongly lignified xylem fibers and phloem fiber sclereids (Figure 4g–i) in OEWOX14 plants. This phenotype was further confirmed by the increased expression levels of transcription factors involved in lignin deposition (MYB85, Figure 5b) and cell wall thickening (SND1 and SND3, Figure S4a; Zhong et al., 2008) in young stems of OEWOX14 plants. wox14 N8514 (a) 0 1 2 3 4 5 6 14 FLC expression Days 0 0.3 0.6 0.9 1.2 1.5 6 14 SOC1 expression Days (d) (c) *** *** (b) 0 5 10 15 20 25 Number of rosette leaves + GA + cold LD *** wox14 N8514

Figure 1. Exogenous gibberellin (GA) or prolonged stratification fully res-cues the wox14 late-flowering phenotype. (a) Morphology of control (N8514) and wox14 plants grown under long-day (LD) conditions for 6 weeks. (b) Leaf number at floral transition of control line (white bar) and wox14 mutant (black bar) plants grown under LD conditions without additional treatment, with exogenous 100 mMGA3 application every 2 days until floral transition (+GA) or with prolonged cold treatment at 4°C for 6 weeks (+cold). Error bars indicate standard deviations (SDs); ***P< 0.001. (c, d) Gene expression levels of FLC (c) and SOC1 (d) relative to TRB3 in 6- and 14-day-old seedlings of wild type (white bar) and wox14 (black bar). Samples were collected 8 h after the beginning of the light period to avoid any circadian rhythm interfer-ence. [Colour figure can be viewed at wileyonlinelibrary.com].

The GA biosynthesis gene GA3ox1 is a downstream target of WOX14 during secondary growth

As mentioned above, increased lignin deposition in vessels and xylem fibers has been previously reported in plants where GA biosynthesis genes are overexpressed, and this in several species, including Arabidopsis (Biemelt et al., 2004; Jeon et al., 2016; Park et al., 2015). Screening for changes in expression levels of GA biosynthesis genes at the stem base indicated that only GA3ox1 transcript levels were clearly deregulated in wox14, with mRNA levels 50% lower than in the control (Figure 5a). GA3ox1 RNA levels were between four and eight times higher in young and mature stems of OEWOX14 lines, respectively (Figures 5a, b and S3c).

Analysis of the GA3ox1:GUS reporter gene indicated that the GA3ox1 promoter is initially active in the pro-cambium, the pith parenchymal cell layers surrounding the xylem pole and the interfascicular cortex (Figure 2d). In inflorescences of 20 cm in height, the staining was largely restricted to the procambium (Figure 2e), extend-ing into the interfascicular cambium region in more mature stems (Figure 2f). Therefore GA3ox1, which is the most abundant GA3ox gene expressed in the stem (Mitchum et al., 2006), has an expression pattern that overlaps with that of WOX14 within the procambium

during stem maturation (Figure 2b, e). Interestingly, anal-ysis of the GA3ox1:GUS reporter gene in the wox14 pri-mary root revealed weaker GA3ox1 promoter activity (Figure S5a), whereas a significant increase in GA3ox1 expression was observed in 10-day-old OEWOX14 seed-lings (Figure S5b). These data further confirm that WOX14 regulates GA3ox1 expression in all tissues where it is expressed.

In Arabidopsis, GA3ox genes are essential for the pro-duction of bioactive GA required for xylem expansion. Indeed, ga3ox1ga3ox2 double mutants have a severe dwarf phenotype and have strongly decreased expression levels of cell wall thickening and lignin deposition tran-scription factors in the stem (Mitchum et al., 2006; Wang et al., 2013). The genetic relationship between WOX14 and GA3ox1 in the stem was evaluated by crossing the OEWOX14 lines with the ga3ox1 null mutant. The ga3ox1 null background fully abolished stem stiffness in OEWOX14. Indeed, OEWOX14 ga3ox1 plants had short soft stems with a stem base diameter similar to that of the single ga3ox1 null mutant (Figures 4a and S6). Cross sec-tions at the stem base showed lower lignification for both the OEWOX14ga3ox1 and single ga3ox1 mutants com-pared with either Col-0 or OEWOX14 plants (Figure 6b–g). Taken together, these data further confirm that GA3ox1 is

ph c xy ph c xy pi 8-9 weeks 6-7 weeks 4-5 weeks (c) (b) (a) ph c xy ph c xy pi pi (e) (f) (d) pi ph c xy ph c xy pi

Figure 2. WOX14 and GA3ox1 expression profiles overlap in cambial cells. Expression of WOX14:GUS (a–c) and GA3ox1:GUS (d–e) reporter genes in sections made at the base (1 cm above the rosette leaves) of 10-cm (a, d), 20-cm (b, e) and 50-cm (c and f) inflorescence stems, respectively. The corresponding growth times are given above each column of pictures. Observations were made on phloroglucinol-stained samples. Abbreviations: c, cambial cells; ph, phloem; pi, pith cells; xy, xylem.

(a) N8514 wox14 50 µm 50 µm 50 µm 50 µm (c) (b) (d) (e) (f) (g) _(h) (i) (j)

Figure 3. Supernumerary vascular bundles (VBs) and hypolignification of the wox14 floral stem are rescued by gibberellin (GA). Vascular development and maturation of the control line N8514 (a, c, e, g, i) and wox14 (b, d, f, h, j) stems. Wall lignification was stained in red using phloroglucinol (a–f, i, j), and xylem tissues were stained in blue–green using toluidine blue (g, h). (a, b) Hand sections of young developing stems made right below the inflores-cence meristem of infloresinflores-cence stems at 25–30 cm in height. (c, d) Hand sections made at the base of inflorescence stems of 2 cm in height in order to observe the early stages of xylem lignification. (e, f, i, j) Hand sections of mature stems made 1 cm above the rosette leaves of inflorescence stems at 25–30 cm in height without exogenous GA treat-ment (e, f), or sprayed with 100 mMGA3 solution (i,

j) every 2 days until floral transition. (g, h) Thin transverse sections of mature stems made 1 cm above the rosette leaves of inflorescence stems at 25–30 cm in height.

a downstream target of WOX14 and plays a major role in promoting cell differentiation during stem maturation. WOX14 overexpression downregulates GA deactivation genes in the inflorescence stem

In Arabidopsis, GA3ox and GA2ox genes have been clearly shown to have a direct role in determining in vivo bioactive GA levels (Alcazar et al., 2005; Mitchum et al., 2006; Oh et al., 2006; Hu et al., 2008; Barboza et al., 2013; Giacomelli et al., 2013). Therefore, we exam-ined the expression of the other stem-specific GA3ox

and GA2ox genes (Mitchum et al., 2006) in the OEWOX14 line.

In wild-type plants, GA3ox2 expression was very weak compared with GA3ox1 (100 times lower) in both young and mature stems (Figure S7a). When WOX14 was overex-pressed, GA3ox2 transcript levels increased in both mature and young stems, although with high variation between biological repeats (Figure S7b). Importantly, our data clearly showed that WOX14 represses the GA catabolism GA2ox genes in stems (Figure 5b, S7c). The most expressed GA2ox gene, GA2ox1, was highly repressed in

(a) 50 µm 50 µm 50 µm 50 µm 100 µm 100 µm (f) (e) (d) (g) (h) (i) Top Base OEWOX14 Col-0 (b) (c) 0 0.5 1 1.5 2

Top Top Mid Mid Base Base

Stem diameter

(mm)

*** ***

Figure 4. Overexpression of WOX14 increases vas-cular lignification and radial growth. (a) Phenotypes of 6-week-old Col-0 (on the left), OEWOX14 (white arrow), OEWOX14/ga3ox1 (yellow arrow) and ga3ox1 (red arrow) plants. (b) Picture showing stem strength of 7-week-old wild type (Col-0) and OEWOX14 plants with no support system. Note that the main stems of the wild type are bent. (c) Mea-surement of the stem diameter (mm) along the stem of 7-week-old Col-0 (white bar) and OEWOX14 (black bar) plants. (d–i) Phloroglucinol-stained hand sections of wild-type (d–f) and OEWOX14 (g–i) stems of 25–30 cm in height. Pictures (e) and (h) are magnifications of (d) and (g), respectively. Sec-tions made at the stem base show a larger region of lignified vascular cells in OEWOX14 (g, h) com-pared with the wild type (d, e). Sections made at the top of the inflorescence meristem show strong and precocious lignification of vessel cells in OEWOX14 (i), compared with the wild type (f). All the plants were grown under long-day (LD) condi-tions. Top and base are defined in Figure S1b. Error bars indicate SDs; ***P< 0.001.

the upper stem of OEWOX14 plants, with its mRNA levels being 70% less than in the control (Figure 5b). This decrease was less important in the basal part of the stem

(Figure S7c). Indeed, in the mature stem of wild-type plants, WOX14 expression is already at its maximal level (Figure S1c), and GA2ox1 gene expression is normally low 8.5 9 9.5 10 10.5 0 0.5 1 1.5 2 Relative expression 0 0.5 1 1.5 WOX14 WOX4 0 0.5 1 1.5 2 2.5 3 3.5 4 (a) *** *** 0 0.5 1 1.5 GA2ox1 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 GA3ox1 (b) Relative expression 0 0.5 1 1.5 *** WOX4 0 0.5 1 1.5 WOX14 0 0.5 1 1.5 2 2.5 MYB85 GA2ox2 0 1 2 3 4 5 6 7 *** *** ** *** *** *** *** *** *** *** * Relative expression GA3ox1

Figure 5. Expression of WOX14, WOX4, GA3ox1, GA2ox1/2 and MYB85 genes in WOX14 and WOX4 mutant and overexpressing backgrounds. (a) Relative tran-script levels of the genes at the base of inflorescence stems of 20–25 cm in height. (b) Relative trantran-script levels of the genes at the top of inflorescence stems of 20–25 cm in height. Normalized gene expression in the mutant (grey bar) and overexpressing (black bar) lines were reported relative to their level in the refer-ence background used, N8514 or Col-0 (white bar). Top and base are defined in Figure S1b. Error bars indicate SDs; ***P< 0.001; **P < 0.01; *P < 0.05.

(Figure S7a). In addition, GA2ox2 expression levels also decreased in the basal part of the OEWOX14 stem (Fig-ure S7c). Taken together, these results suggest that one function of WOX14 is to increase the production of bioac-tive GA by upregulating the GA biosynthesis GA3ox genes and repressing the GA deactivation GA2ox genes.

WOX14 overexpression increases bioactive GA content in the inflorescence stem

To evaluate the effects of changes in GA metabolism gene expression, we quantified the different forms of bioactive GA in the stem. In agreement with the molecular data described above, an increase in the overall bioactive GA content was found in the OEWOX14 stem (Figure 6a). GA4,

which is considered to be the most active form of GA in Arabidopsis (Xu et al., 1997; Eriksson et al., 2006), remained constant; however, we found higher levels of its inactive form (GA34), suggesting that GA4 deactivation is

increased to maintain GA homeostasis when its biosynthe-sis is induced. GA1was the most abundant active form in

the stem and its levels were twice as high in OEWOX14 plants, although its inactive form (GA8) also increased.

Hence, our data further confirm the role of WOX14 in pro-moting the production of bioactive GA required for cell dif-ferentiation.

WOX4 does not act redundantly with WOX14 in promoting cell differentiation during stem maturation A previous study has shown that WOX14 acts redundantly with WOX4 to regulate cambial cell proliferation (Etchells et al., 2013). Therefore, we investigated a possible redun-dancy of both genes in regulating GA activation and deac-tivation genes for cell differentiation.

WOX4 expression in the wild type is constant through-out stem development within the fascicular vascular meris-tem, whereas WOX14 is more highly expressed in mature tissues (Etchells et al., 2013; Figure S1b). Analysis of WOX4 expression in the wox14 mutant revealed that in the stem base of 7-week-old plants mRNA levels were higher (Figure 5a). No major changes in WOX4 expression were found in the OEWOX14 line (Figure 5a), however, indicat-ing that WOX4 is not involved in the OEWOX14 pheno-types.

In wox4 and wox4 wox14 mutants, GA3ox1 expression at the stem base was lower than in the wox14 single mutant (Figure 5a), suggesting that WOX4 could also pro-mote GA3ox1 expression; however, because WOX14 expression at the stem base is extremely low in wox4 (Fig-ure 5a), the contribution of each WOX gene in controlling GA3ox1 expression is difficult to evaluate.

As WOX4 expression is more specific to the upper part of the stem, we expected a clearer indication of the effect of its mutation in this region. In addition, we also used a previously described WOX4 overexpressing line (hereafter OEWOX4, Figure S3d; Suer et al., 2011) to further investi-gate whether WOX4 plays a role in GA3ox and GA2ox reg-ulation, and in vascular cell differentiation. In the young stem, WOX14 expression was only slightly decreased in wox4 but was 50% lower in the OEWOX4 line (Figure 5b). Importantly, GA3ox1 expression levels did not change

50 µm 50 µm 50 µm 50 µm 50 µm 50 µm 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 GAs content (pg mg –1 DW) GA4 GA34 GA1 GA8 (b) (a) (c) (e) (d) (g) (f)

Figure 6. Effect of WOX14 overexpression on gibberellin (GA) production and role of GA3ox1 on the vascular lignification phenotype. (a) Dosage of bioactive GA and its inactivated products in wild-type (white bar) and OEWOX14 (black bar) stems. (b–g) Phloroglucinol-stained sections of the wild-type Col-0 (b), OEWOX14 (c), ga3ox1 (d, e) and OEWOX14 ga3ox1 (f, g) stem bases of 6-week-old plants. Top and base are defined in Figure S1b. Sections (70-mm thick) were obtained using a Vibratome. All plants were grown under long-day (LD) conditions.

significantly in either the WOX4 mutant or the overex-pressing lines, whereas an almost fourfold increase was observed in the overexpressor of WOX14 (Figure 5b). In addition, the GA3ox2 level was almost 50% lower in OEWOX4 (Figure S7b). Altogether, our data indicate that, unlike WOX14, WOX4 appears to inhibit the expression of GA activating genes.

The genetic link between WOX4 and the GA deactivation genes in the upper stem is less clear. GA2ox1 expression was reduced in OEWOX4 plants but was not affected in wox4, and was even reduced in wox4 wox14 (Figure 5b). GA2ox2 mRNA levels were reduced in OEWOX4 plants, and its levels were 50% higher in wox4 and between three and six times higher in wox4 wox14 suggesting that the two WOX genes act redundantly to repress GA2ox2 expression.

Therefore, overall changes in GA3ox and GA2ox mRNA levels within the different genetic backgrounds do not clearly indicate how WOX4 affects bioactive GA synthesis in stems. Nevertheless, the expression levels of MYB85, a lignification reporter gene, did not vary in the OEWOX4 line (Figure 5b), indicating that, unlike WOX14, WOX4 overexpression is not sufficient to promote vascular cell lignification.

DISCUSSION

WOX14, GA and the control of cell differentiation

The control of cell differentiation within the cambial zone relies on both genetic and hormonal pathways. WOX genes and GAs are important players in the control of cam-bial activity; however, the molecular mechanisms by which they interact are still poorly understood.

Bioactive GA accumulates preferentially within xylem precursors during wood formation to promote the differen-tiation of xylem cell initials and lignin biosynthesis (Israels-son et al., 2005; Mauriat and Moritz, 2009). Our results indicate that the level of bioactive GA required for vascular cell differentiation is increased by WOX14 within the VBs as: (i) wox14 mutants have many phenotypes that suggest a deficit in GA content or signaling (Figures 1, 3); (ii) using an overexpressing approach, we demonstrated that WOX14 positively regulates the expression of GA3ox genes and negatively regulates the expression of GA2ox genes (Figure 5b); (iii) the GA1 level is increased in the

stem (Figure 6a); and (iv) GA3ox1 is a downstream target of WOX14 for regulating vascular cell differentiation (Fig-ure 6b). This is consistent with weaker lignification of the xylem in wox14 (Figure 3), and increased xylem formation and precocious tissue lignification when WOX14 is overex-pressed (Figures 4g–I, 5b).

According to our data, WOX14 is involved in promoting cell differentiation rather than cell proliferation within the cambial zone. This contrasts with a previous study that

suggested that WOX14 functions redundantly with WOX4 in promoting cell proliferation (Etchells et al., 2013).

Indeed, both WOX4 and WOX14 mRNA accumulate in cambial cells during stem maturation; however, WOX4 overexpression did not result in greater cambial activity or radial growth (Hirakawa et al., 2010b), as is observed with WOX14. Interestingly, their mutation or overexpression indicated that they indirectly affect the expression of each other (Figure 5). WOX4 RNA levels were higher in the stem base of 7-week-old wox14 plants, and an analysis con-ducted in samples of 6-week-old plants indicated that this increase arose from a longer persistance of WOX4 in the wox14 background (Figure S4b). We also noticed that WOX14 expression was also reduced when WOX4 was overexpressed or mutated (Figure 5). Considering that WOX4 function is to promote the proliferation of stem cells (Hirakawa et al., 2010b; Suer et al., 2011) and that WOX14 accumulates in these cells during stem maturation, it can be hypothesized that WOX14 mRNA is lower in wox4 as cambial cell numbers are reduced, whereas WOX14 mRNA accumulation, and consequently vascular differentiation, is delayed when WOX4 is overexpressed. Conversely, WOX4 expression might persit because vascular differentiation is impaired in wox14.

Although GA1is thought to be less active than GA4, and

is present at low levels in Arabidopsis vegetative tissues (Talon et al., 1990; Yamaguchi, 2008; Nomura et al., 2013), we found it to be the most abundant bioactive form in the Arabidopsis stem (Figure 6a), similarly to what has been previously found in developing siliques (Varbanova et al., 2007). GA3ox genes are essential for the synthesis of bioactive GA in Arabidopsis, but their overexpression alone is not sufficient to increase bioactive GA production, presumably because of a strong negative feedback regula-tion on the endogenous GA3ox promoters (Phillips, 2004; Mitchum et al., 2006; Radi et al., 2006; Wang et al., 2013). It is known that GA homeostasis is a tightly regulated pro-cess during plant development where bioactive GAs nega-tively regulate their own production by activating GA2ox genes and repressing GA3ox genes (Itoh et al., 2008; Hed-den and Thomas, 2012). Therefore, to be effective in induc-ing xylem cell differentiation, a signal has to control both the induction of GA3ox and the repression of GA2ox gene expression in order to increase the bioactive GA content in the Arabidopsis stem.

Our analyses of young stem tissues indicate that WOX4 does not act on GA3ox and GA2ox genes in a way that pro-motes bioactive GA accumulation and vascular differentia-tion in the stem (Figure 5b and S4). This is supported by our finding that expression levels of MYB85, the lignin biosynthesis marker gene, remain unchanged when WOX4 is overexpressed. Our data rather suggest that the expres-sion of both GA3ox and GA2ox genes are repressed in cells expressing WOX4, and that only WOX14

simultaneously promotes GA activating genes and represses GA deactivation genes in the stem to increase the production of bioactive GA required for cell differentia-tion.

Whether the bioactive GA promoted by WOX14 could also modulate vascular cell proliferation in Arabidopsis remains unknown. In Nicotiana tabacum (tobacco), the function of bioactive GA in cambial cells was clearly described as being a promoter of xylem fiber differentia-tion, although cambial cell proliferation and differentiation seem to be dependent on the synergic action of both auxin and gibberellin during secondary growth (Itoh et al., 1999; Biemelt et al., 2004; Dayan et al., 2012). Interestingly, the CLE41/PXY signal that has been shown to positively regu-late WOX14 and cell proliferation in Arabidopsis (Hirakawa et al., 2010b; Ji et al., 2010), has also been shown to be a positive regulator of cambial activity and wood formation in hybrid aspen (Etchells et al., 2015). Because the PXY/ WOX4 pathway has been suggested to stimulate the auxin responsiveness in the cambium (Suer et al., 2011), we can-not exclude that WOX14 could also stimulate the auxin responsiveness of cambium cells. Hence, it is conceivable that WOX14 could promote cell proliferation through GA biosynthesis, depending on the cellular context. Further characterization of the interaction between the two WOX genes and the GA pathway with regards to the control of cell production should give a better understanding of the importance of GA signaling in controlling cambial cell activity in Arabidopsis.

WOX14 functional evolution

Both WOX4 and WOX14 accumulate in cambial cells; how-ever, the accumulation of WOX14 in the stem base sug-gests that WOX14 is more important during secondary growth than during primary VB development. Furthermore, WOX14 has been shown to be epistatic to PXY, whereas WOX4 is not (Etchells et al., 2013). Comparative analysis of the wox4 pxy, wox14 pxy and wox4 wox14 double mutants have also indicated that their regulation differs in Ara-bidopsis (Hirakawa et al., 2010b; Ji et al., 2010). As many signaling pathways with a WOX gene also include a CLE/ PXY-like ligand/receptor (Barra-Jimenez and Ragni, 2016), we can propose a regulatory model in Arabidopsis where an additional xylem promotor signal (CLE/PXY-like) also controls the expression of WOX14 to promote GA produc-tion for optimizing vascular cell differentiaproduc-tion during wood formation, and overcomes the CLE41/PXY/WOX4 sig-nal implicated in cambial stem cell proliferation (Figure 7).

Unlike WOX4, WOX14 is not a member of the recent WUS clade, but belongs to the most ancestral WOX clade. Although both Arabidopsis members of the latter clade, WOX13 and WOX14, are expressed in VBs (Deveaux et al., 2008), the phenotypes of wox14 single mutants suggest that WOX13 and WOX14 functions are not redundant

regarding the control of GA metabolism. In addition, WOX14 is found only in Brassicaceae, and it can be sug-gested that GA regulation could result from a neo-functio-nalization of the WOX14 paralog; however, we cannot exclude that sub-functionalization has occurred in this clade, leading to the regulation of bioactive GA production by WOX13 in other species. Further characterization of WOX13 genes in Arabidopsis and in a range of species, including trees, will be necessary to better understand the functional evolution of the two paralogs in relation to the GA pathway.

Interestingly, a recent study in Physcomitrella patens, which has only WOX13-like genes, has suggested that these genes are involved in the initiation of cell growth, and positively regulate cell wall loosening genes during stem cell formation (Sakakibara et al., 2014). Although putative paralogs of GA biosynthesis genes exist in moss, P. patens lacks any response to exogenous GA, and a DELLA mutation does not affect its cell growth (Yasumura et al., 2007). Therefore, control of GA metabolism by WOX members of the ancestral group probably arose after the divergence of bryophytes from other land plants.

Vascular cell differentiation GA9/20 GA1/4 GA8/34

Ga3ox Ga2ox WOX 14 WOX4 PXY CLE41p PXY-like? CLE? Vascular cell proliferation

X

Figure 7. A model of vascular meristem activity integrating WOX and gib-berellin (GA) signaling. A xylem-promoting signal (CLE/PXY-like?) leads to WOX14 accumulation in the stem, which promotes GA biosynthesis and induces vascular cell differentiation during secondary growth. WOX4 may act as a repressor of GA metabolism genes, leading to the maintenance of vascular stem cells.

EXPERIMENTAL PROCEDURES Plant material

The wox14 line pst13645 was obtained from the RIKEN BioRe-source Center (BRC, http://en.brc.riken.jp), Japan. Donor line N8514 ecotype Nossen (No) was chosen as the reference line in this background. Seeds of wox4-1/GABI462G01 (N376577), ga3ox1 (N6943) and GA3ox1:GUS ecotype Col-0 (N16356) were obtained from the Nottingham Arabidopsis Stock Center (NASC, http://arab idopsis.info). The wox4-1 mutant and WOX4 overexpressing lines have been described previously (Hirakawa et al., 2010b; Suer et al., 2011). In order to generate the other lines used in this study, the wox14-pst13645 allele was introgressed into the Col-0 back-ground through backcrossing using microsatellite marker-assisted selection. After the fourth backcross, wox14 plants highly enriched in the Col-0 genome were selected as a parent to generate the double mutants and the reporter lines. Wild-type Col-0 plants were used as a control.

Growth conditions

Seeds for in vitro culture were surface-sterilized and stratified on half-strength MS medium, with 1% sucrose and 0.7% agar, at 4°C for 2 days, then grown in long-day (LD) conditions at 20°C. Plants used for transcript quantification from inflorescence stems were directly sown in soil and grown in a growth chamber under LD conditions at 20°C. Observation of root development was per-formed on plants grown in soil under short-day (SD) conditions at 20°C.

For transgenic plant screening, seedlings were selected in vitro on 10 mg mL
1phosphinothricin (PPT).

GA3 (Duchefa Biochemie, http://www.duchefa-biochemie.com) treatment was applied to 7-day-old soil-grown plants at a concen-tration of 100 mM. Plants were sprayed with GA3 solution twice a week until bolting.

Cold treatment was performed by incubating seeds sown on half-strength MS agar medium with 1% sucrose for 6 weeks at 4°C in the dark. Seedlings were subsequently transferred to soil and grown under LD conditions.

Production of transgenic lines

For the complementation assay, an Arabidopsis thaliana WOX14 cDNA driven by a previously characterized 1-kb fragment of the WOX14 promoter was produced (Deveaux et al., 2008). To gener-ate the OEWOX14 line, WOX14 cDNA was amplified by RT-PCR using specific primers (50 -GGATCCAAGGAAAAGGAGAAAAG-CAAAGA-30, 50-CGAATTCT GTCACACACAAACACACACA-30) and cloned under the control of the 35S CaMV promoter. The full chi-meric gene was transferred to the pPF11 binary vector derived from pPZP100 (Hajdukiewicz et al., 1994) before plant transforma-tion. The WOX14:GUS line in Col-0 and transgenic lines were obtained as described previously (Deveaux et al., 2008). The OEWOX14 transgene copy was genotyped using primers that matched the 35S promoter and the cDNA sequence respectively: 50-ACGCACAATCCCACTATCCTTCG-30 and 50-AGAATTCTTGAGG CTGCTTTCGTTTGG-30.

RNA extraction, RT and real time PCR

Total RNA was extracted from 6- and 14-day-old seedlings, and from 1.5-cm sections of inflorescence stems using the Qiagen RNeasy plant extraction kit and DNAseI treatment according to the manufacturer’s instructions. RNA (1–5 mg) were reverse

transcribed with the Superscript II reverse transcriptase from Invitrogen (now ThermoFisher Scientific, http://www.thermofishe r.com). Quantitative PCR was performed on a Roche Light Cycler 480 using the SYBER green PCR mix (Roche, http://www. roche.com). At3g49850 (TRB3) was used to normalize RNA input, except for the wox4 wox14 double mutant where At4g05320 (UBQ10) was used. Alternatively, relative fold induc-tion was used to compare normalized gene expression relative to a reference sample. Each experiment was performed on at least two biological samples and all data shown included tech-nical repeats. Error bars refer to both techtech-nical and biological standard deviations. P values were determined using the bilat-eral Student’s t-test. See Table S1 for oligonucleotide sequences.

Staining and observations

Beta-glucuronidase (GUS) staining was performed as previously described (Bertrand et al., 2003) on seedlings or stem sections. Phloroglucinol staining (Sigma-Aldrich, http://www.sigmaaldric h.com) was also performed and combined with GUS staining when needed. Hand-cut or 70-mm-thick vibratome sections were mounted on slides in HCG (8 g Chloral hydrate, 2 mL Glycerol 50%, 1 mL water) for observation using a Nikon Multizoom AZ100 macroscope (Nikon, http://www.nikon.com). Image acquisition was achieved with a DS-Ri1 camera and

NIS-ELEMENTS D(Nikon). Toluidine staining was performed on thin sections of samples that were embedded in Paraplast Plus (Sherwood Medical, St. Louis, MO, USA).

GAs quantification

GA content was measured in stems of 6-week-old plants grown under LD conditions. After removal of the first 3 cm from the inflo-rescence tip and the first 2 cm from the rosette leaves, the remain-ing stem samples were ground and freeze-dried. Quantification was carried out by the Laboratory of Growth Regulators, Czech Republic, as described previously (Urbanova et al., 2013) on three replicates of 10 mg dry weight (DW) samples.

ACCESSION NUMBERS

At1G20700 (WOX14), At1G46480 (WOX4), At2G45660 (AGL-20/SOC1), At5G10140 (AGL-25/FLC), At1G15550 (GA3ox1), At1G80340 (GA3ox2), TRB3 (AT3G49850), GA2ox1 (AT1G78440), GA2ox2 (AT1G30040), UBQ10 (AT5G53300). ACKNOWLEDGEMENTS

We thank Alain Lecharny, Moussa Benhamed, Beatriz Goncßalvez and Vincent Thareau for scientific discussions and/or help with the bioinformatic strategy. We acknowledge Prof. Thomas Greb (COS, Germany) for providing us with the OEWOX4 line, Dr Danuse Tarkowska for carrying out the GA quantification and the Institute’s technical team for their support, especially Sophie Mas-sot. We thank Catherine Damerval for careful reading and sugges-tions, Sarah Rosa and Marjorie Guichard for their contributions during their internship, and Claire Toffano-Nioche for her help in obtaining a grant from the PPF13 (PPF Bioinformatique et BioMathematique) fund. This work was funded by the CNRS and the Universite Paris Sud. The english text was edited by Helene Citerne.

CONFLICTS OF INTEREST

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online ver-sion of this article.

Figure S1. wox14 phenotype, promoter activity and stem expres-sion.

Figure S2. wox14 vascular bundle (VB) and floral transition com-plementation assays.

Figure S3. GA3ox1, WOX14 and WOX4 transcript levels in the overexpressing lines.

Figure S4. SND1, SND3 and WOX14 expression in 6-week-old stems.

Figure S5. GA3ox1 activity relative to the WOX14 genotype in seedlings.

Figure S6. Effect of the ga3ox1 background on the OEWOX14 phe-notype.

Figure S7. Relative expression levels of GA3ox and GA2ox in young and mature stems.

Table S1. Oligonucleotides used for quantitative RT-PCR. REFERENCES

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