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. 2022 Jun 29;34(10):3737–3753. doi: 10.1093/plcell/koac188

WOX family transcriptional regulators modulate cytokinin homeostasis during leaf blade development in Medicago truncatula and Nicotiana sylvestris

Hui Wang 1,2,✉, Xue Li 3, Tezera Wolabu 4, Ziyao Wang 5, Ye Liu 6, Dimiru Tadesse 7, Naichong Chen 8, Aijiao Xu 9, Xiaojing Bi 10, Yunwei Zhang 11, Jianghua Chen 12, Million Tadege 13,✉
PMCID: PMC9516142  PMID: 35766878

One-sentence summary

Modern clade WOX member LAM1 represses NsCKX3 expression, while the intermediate clade member NsWOX9 activates it to modulate cytokinin homeostasis during leaf blade outgrowth in Nicotiana sylvestris.

Abstract

The plant-specific family of WUSCHEL (WUS)-related homeobox (WOX) transcription factors is key regulators of embryogenesis, meristem maintenance, and lateral organ development in flowering plants. The modern/WUS clade transcriptional repressor STENOFOLIA/LAMINA1(LAM1), and the intermediate/WOX9 clade transcriptional activator MtWOX9/NsWOX9 antagonistically regulate leaf blade expansion, but the molecular mechanism is unknown. Using transcriptome profiling and biochemical methods, we determined that NsCKX3 is the common target of LAM1 and NsWOX9 in Nicotiana sylvestris. LAM1 and NsWOX9 directly recognize and bind to the same cis-elements in the NsCKX3 promoter to repress and activate its expression, respectively, thus controlling the levels of active cytokinins in vivo. Disruption of NsCKX3 in the lam1 background yielded a phenotype similar to the knockdown of NsWOX9 in lam1, while overexpressing NsCKX3 resulted in narrower and shorter lam1 leaf blades reminiscent of NsWOX9 overexpression in the lam1 mutant. Moreover, we established that LAM1 physically interacts with NsWOX9, and this interaction is required to regulate NsCKX3 transcription. Taken together, our results indicate that repressor and activator WOX members oppositely regulate a common downstream target to function in leaf blade outgrowth, offering a novel insight into the role of local cytokinins in balancing cell proliferation and differentiation during lateral organ development.

IN A NUTSHELL.

Background: Members of the plant-specific WUSCHEL (WUS)-related homeobox (WOX) family of transcription factors are key regulators of meristem maintenance and lateral organ development. We have shown previously that STENOFOLIA (STF) from Medicago truncatula and LAMINA1 (LAM1) from Nicotiana sylvestris function as repressors, while the intermediate member WOX9 behaves as an activator. Ectopic expression of WOX9 in both stf and lam1 mutant backgrounds exacerbates their respective phenotypes, while silencing of WOX9 by RNA interference in each mutant partially rescues both the stf and lam1 mutant phenotypes, indicating that WOX9 functions antagonistically to STF/LAM1 in leaf blade development. However, the molecular mechanism of this antagonistic regulation remains unknown.

Question: We aimed to identify the common targets of STF/LAM1 and WOX9 and their underlying mechanism during leaf blade outgrowth.

Findings: We demonstrated the antagonistic regulation of active cytokinin contents through regulating cytokinin degradation by two evolutionarily distinct groups of WOX transcription factors: repression by LAM1/modern clade and activation by WOX9/intermediate clade. We also determined that LAM1 physically interacted with NsWOX9, and that both LAM1 and NsWOX9 directly bind to the same cis-elements of the cytokinin oxidase 3 (NsCKX3) promoter in vivo to control local cytokinin levels in balancing the cell proliferation and differentiation to regulate leaf blade expansion.

Nest steps: As we noticed that the phenotypic rescue the stf/lam1 mutant was not complete, further experiment should focus on the identification of the additional components needed to fully rescue the stf/lam1 phenotypes.

Introduction

Members of the WUSCHEL (WUS)-related homeobox (WOX) family are plant-specific transcription factors that belong to the homeodomain (HD) superfamily, named after the founding member of the group, Arabidopsis (Arabidopsis thaliana) WUS (Mayer et al., 1998). WOX members regulate a variety of plant developmental programs including shoot and root meristem maintenance (Schoof et al., 2000; Wu et al., 2005; Sarkar et al., 2007), embryo development (Ueda et al., 2011), vascular patterning (Hirakawa et al., 2010), and the development of lateral organs (Nardmann et al., 2004; Dai et al., 2007; Lin et al., 2013). Based on their evolutionary appearance in the plant kingdom, WOX family members have been divided into three clades: the modern or WUS clade; the intermediate or WOX9 clade; and the ancient or WOX13 clade (Haecker et al., 2004; van der Graaff et al., 2009). While WUS clade members primarily function as transcriptional repressors, WOX9 and WOX13 clade members behave as transcriptional activators in protoplast dual luciferase (LUC) assays (Lin et al., 2013). Multiple orthologs belonging to the WUS clade regulate leaf blade and flower petal development in their respective species: STENOFOLIA (STF) in barrel medic (Medicago truncatula) (Tadege et al., 2011), LAMINA1 (LAM1) in woodland tobacco (Nicotiana sylvestris) (McHale, 1992), MAEWEST in petunia (Petunia × hybrida) (Vandenbussche et al., 2009), LATHYROIDES in pea (Pisum sativum) (Zhuang et al., 2012), SlLAM1 in tomato (Solanum lycopersicum) (Wang et al., 2021), and WOX1 in Arabidopsis (Nakata et al., 2012). STF/LAM1/WOX1 are specifically expressed at the juxtaposition of the adaxial and abaxial domains of the leaf primordia, the so-called “middle domain,” and regulate lamina expansion by promoting cell proliferation (Tadege et al., 2011; Nakata et al., 2012). Despite differences in the strength of leaf blade phenotype exhibited by each respective mutant in the three species (nearly naked midrib in lam1, strong prevention of lateral expansion in stf, and modestly narrow blade only in the wox1 prs [pressed flower] double mutant), the genes STF, LAM1, and WOX1 are functionally interchangeable, and promote blade outgrowth via a transcriptional repression mechanism (Lin et al., 2013; Zhang et al., 2014). The STF/LAM1/WOX1 subclade has two conserved EAR-like (ERF-associate amphiphilic repression motif) repressive motifs: the WUS box, which is common to all WUS clade members, and the STF box, which serves as a diagnostic of the subclade (Tadege et al., 2011; Zhang et al., 2014). The WUS box and STF box of M. truncatula STF recruit the transcriptional co-repressor TOPLESS (MtTPL) to exert transcriptional repression in leaf development analogously to Arabidopsis WUS recruiting TPL for shoot apical meristem (SAM) maintenance (Kieffer et al., 2006; Zhang et al., 2014; Dolzblasz et al., 2016; Meng et al., 2019).

In contrast to WUS clade members, WOX9 clade members lack the repression motifs, endowing them with a transcriptional activation function, with WOX9 possessing the strongest apparent activation activity of all WOX9 and WOX13 clade members in Arabidopsis (Lin et al., 2013). WOX9 is a versatile transcriptional regulator with different mutant phenotypes in several species that illustrate the involvement of this protein in many developmental programs including embryo polarity patterning (Skylar et al., 2010; Ueda et al., 2011; Zhu et al., 2014), SAM maintenance (Wu et al., 2005, 2007), inflorescence architecture (Rebocho et al., 2008; Hendelman et al., 2021), and leaf blade development (Kyo et al., 2018; Wolabu et al., 2021). In Arabidopsis, the WOX9 loss-of-function stimpy (stip) mutant is arrested at the seedling stage, underscoring the requirement for WOX9 function in meristem maintenance (Wu et al., 2005; Skylar et al., 2010). The evergreen mutant in petunia and various targeted alleles and cis-element analysis in tomato have clearly demonstrated that WOX9 has a profound effect on inflorescence architectures (Lippman et al., 2008; Rebocho et al., 2008; Hendelman et al., 2021).

Crosstalk between WOX family members has already been demonstrated in plants. STIP/WOX9 positively regulates WUS in maintaining cell pluripotency and proliferation in apical meristematic tissues in Arabidopsis (Wu et al., 2005). We recently reported that MtWOX9 and NsWOX9, involved in the regulation of the abaxial cell fate during leaf blade development, are directly repressed by STF or LAM1 in M. truncatula and N. sylvestris, respectively (Wolabu et al., 2021). These results indicate that WOX family members closely interact with each other to mediate cell proliferation and differentiation during lateral organ development.

Phytohormones also play important roles in cell proliferation and differentiation during multiple stages of plant development. A close relationship has been reported between WOX family members and the phytohormone cytokinin. The WUS clade members Arabidopsis WUS and M. truncatula HEADLESS maintain SAM activity in the stem cell niche by activating cytokinin signaling through the repression of type-A ARABIDOPSIS RESPONSE REGULATOR (ARR) gene expression (Leibfried et al., 2005; Meng et al., 2019). Arabidopsis WOX9 is also involved in photomorphogenesis as an effector of the cytokinin signaling pathway (Skylar et al., 2010; Skylar and Wu, 2010). Cytokinins are key growth regulators involved in a variety of plant developmental events including cell proliferation (Werner et al., 2003; Reid et al., 2016). The levels of active cytokinins in plant tissues are controlled by a balance between their biosynthesis, degradation, translocation, and inactivation (Sakakibara, 2006). It is unclear which of these steps are controlled by STF/LAM1 and/or MtWOX9/NsWOX9. We recently reported that ectopic expression of M. truncatula or N. sylvestris WOX9 exacerbates the stf and lam1 mutant phenotypes, respectively (Lin et al., 2013; Wolabu et al., 2021), suggesting that the WOX members STF and WOX9 function antagonistically in leaf blade development. However, the molecular mechanism of this antagonistic regulation is completely unknown.

Here we demonstrate that STF/LAM1 and MtWOX9/NsWOX9 have a common downstream target, CYTOKININ OXIDASE3 (CKX3) that is transcriptionally regulated in opposite directions. While LAM1 directly repressed NsCKX3 to activate cytokinin signaling, NsWOX9 activated NsCKX3 expression by direct binding to multiple regions of its promoter to catabolize cytokinins, thus decreasing cytokinin signaling. We also discovered that LAM1 and NsWOX9 bind to the same cis-elements in the NsCKX3 promoter. Overexpression of NsCKX3 in the lam1 mutant exacerbated the lam1 mutant phenotype with additional defects in leaf length similar to those seen upon ectopic expression of MtWOX9 in lam1, while suppression of NsCKX3 partially rescued the lam1 mutant and the WOX9 overexpression leaf blade phenotypes. Our results suggest that NsCKX3 is the direct common target of LAM1-mediated transcriptional repression and NsWOX9-mediated transcriptional activation, which is centered around maintaining cytokinin homeostasis for cell proliferation and differentiation during leaf blade development.

Results

Transient WOX9 expression alters CKXs expression levels in M. truncatula and N. sylvestris

We previously showed that the WOX9 transcript levels are upregulated in stf, lam1, and wox1 prs mutants, and ectopic expression of MtWOX9 in M. truncatula and tobacco exacerbates both stf and lam1 mutant phenotypes with additional defects in the proximodistal axis (Supplemental Figure S1; Wolabu et al., 2021), while reducing NsWOX9 expression levels partially rescues the lam1 mutant phenotype (Wolabu et al., 2021). These results suggested that MtWOX9 functions antagonistically to STF/LAM1 in leaf blade development, and is negatively regulated by STF. To confirm the transcript levels and pattern of MtWOX9 in the stf mutant, we performed RNA in situ hybridization. In longitudinal section of the vegetative apex, MtWOX9 was not expressed at the center but in the peripheral region and at the position of organ separation between the meristem proper and emerging leaf primordia, and at the base of established primordia, with no differences between the wild-type R108 and the stf mutant (Figure 1, A and B). However, at a later stage of leaf development (P7), we detected a stronger signal from MtWOX9 transcripts in the stf mutant than in R108, and the signal expanded to the middle domain in the stf mutant compared to R108 (Figure 1, C and D), confirming that MtWOX9 is repressed by STF in the middle domain of the leaf primordia where STF is expressed. This result was consistent with the previous report that STF directly binds to the MtWOX9 promoter and represses its transcription (Wolabu et al., 2021). Note that the so-called “middle domain” is a one-to-two cell-layer tissue at the juxtaposition of the adaxial and abaxial domains of the leaf blade including the margin and middle mesophyll that marks the STF/WOX1 expression pattern and that governs blade lateral expansion through promotion of cell proliferation (Tadege et al., 2011; Nakata et al., 2012; Nakata and Okada, 2012). To identify the downstream molecular events of MtWOX9 and STF/LAM1 interaction, we generated M. truncatula transgenic lines harboring a transgene encoding the yellow fluorescent protein (YFP) fused to the glucocorticoid receptor (YGR) and to MtWOX9. First, we confirmed that the phenotypes of 35S:YGR-MtWOX9 lines treated with the inductive reagent, dexamethasone (DEX), develop leaf blade phenotypes similar to those of MtWOX9 ectopic expression lines (Supplemental Figure S2). We then performed transcriptome deep sequencing (RNA-seq) analysis of folded leaves from three independent lines harvested 3 h after treatment with DEX and the protein synthesis inhibitor cycloheximide (CHX). We identified 40 significantly upregulated genes and 10 downregulated genes with a Log2(fold-change) of at least 2 in the lines treated with DEX + CHX compared to uninduced control plants treated with CHX alone (Figure 2; Supplemental Figure S3A). Because MtWOX9 functions as a transcriptional activator in leaf blade expansion, we considered the upregulated genes as potential direct targets. We combined this data together with the Affymetrix gene chip data on the stf mutant (Tadege et al., 2011) to identify genes highly responsive to the activity of both MtWOX9 and STF. This analysis identified, among others, two putative cytokinin oxidase/dehydrogenase (CKX) genes Medtr4g126150 and Medtr2g039410, which were upregulated four- and nine-fold, respectively, in DEX + CHX-treated MtWOX9 lines compared to CHX-treated controls (Supplemental Figure S3B). We selected these two genes for further analysis because both STF and Arabidopsis WOX9 were shown to mediate cytokinin effects in transgenic rice and Arabidopsis, respectively (Skylar et al., 2010; Wang et al., 2017). To test whether MtWOX9 regulated Medtr4g126150 and Medtr2g039410 transcript levels, we constructed transgenic lines with more (overexpression) or less (by RNA interference [RNAi]) MtWOX9 transcripts. The leaves of MtWOX9-RNAi lines were comparable to those of the non-transgenic control, while 35S:MtWOX9 lines exhibited narrow leaves closely associated with their transcript abundance (Supplemental Figure S4, A–C). The transcript levels of Medtr4g126150 and Medtr2g039410 were significantly downregulated in MtWOX9-RNAi lines and strongly increased (at least 30-fold) in 35S:MtWOX9 overexpression lines (Supplemental Figure S4D). Transcript levels for these two genes were also significantly upregulated in the 35S:MtWOX9 line in the stf mutant background, which showed severe phenotypes (Supplemental Figures S1S5). These results indicate that these two CKX genes are candidate targets of MtWOX9 transcriptional activation in M. truncatula.

Figure 1.

Figure 1

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RNA in situ hybridization of MtWOX9 in the vegetative shoot apex and leaf primordia of M. truncatula R108 and the stf mutant. A and B, MtWOX9 expression in the vegetative shoot apex of 3-week-old wild-type R108 (A) and stf mutant (B) viewed in longitudinal sections. C and D, MtWOX9 expression in the P7 stage of leaf development in wild-type R108 (C) and stf mutant (D), viewed in longitudinal sections. Asterisk indicates the SAM. P1, P2, and P7 indicate different developmental stages of leaf primordia. AD, adaxial; AB, abaxial; St, stipule. Scale bars = 50 µm in (A and B) and 100 µm in (C and D).

Figure 2.

Figure 2

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Heatmap of representative differentially expressed genes in MtWOX9 induced expression lines in M. truncatula. Transcriptome deep sequencing (RNA-seq) analysis of folded leaves at the P6 stage in three independent transgenic plants after 3 h of induction with 10-µM DEX. Three-week-old T2 transgenic plants harboring the 35S:YGR-MtWOX9 transgene were sprayed with either 10-µM CHX alone as control or 10-µM CHX and 10-µM DEX.

To determine whether overexpression of MtWOX9 in N. sylvestris induced NsCKX genes expression as in M. truncatula, we isolated seven NsCKXs and examined their transcript levels by reverse transcription-quantitative real time PCR (RT-qPCR). We observed that three out of the seven NsCKX genes (NsCKX1L, NsCKX3, and NsCKX3L1) are expressed at considerably higher levels in the lam1 mutant and in MtWOX9 transgenic lines ectopically expressing MtWOX9 from the STF promoter (Figure 3A). Phylogenetic analysis showed that NsCKX3 and NsCKX3L1 are the closest homologs to Medtr2g039410 (MtCKX3A) and Medtr4g126150 (MtCKX3B) (Supplemental Figure S6). Because MtCKX3A and MtCKX3B were upregulated in MtWOX9 overexpression lines, and NsCKX3 and NsCKX3L1 are the closest homologs showing a similar response to STF/LAM1 mutation and MtWOX9 ectopic expression, we investigated these two genes in six independent N. sylvestris lines ectopically expressing MtWOX9 from the STF promoter. The third gene, NsCKX1L, was not further considered as it belongs to a phylogenetically separate clade, and its expression activated by 35S:YGR-NsWOX9 only in one out of four lines. Consistent with the effect of MtWOX9 overexpression in M. truncatula, we observed that both NsCKX3 and NsCKX3L1 expression is significantly upregulated in STFpro:MtWOX9 N. sylvestris lines (Supplemental Figure S7). To validate that NsCKX3 and NsCKX3L1 are the direct targets of STF/LAM1 and/or WOX9 in M. truncatula and N. sylvestris, we employed a glucocorticoid receptor (GR) induction system in the presence of CHX. Accordingly, we treated 35S:YGR-MtWOX9 lines and 35S:YGR-STF lines in M. truncatula and 35S:YGR-LAM1 and 35S:YGR-NsWOX9 lines in N. sylvestris with DEX and CHX or CHX alone as control. We then collected leaves 3 h later and measured transcript levels of NsCKX3, NsCKX3L1, MtCKX3A, and MtCKX3B by RT-qPCR in the respective lines. We determined that these two pairs of genes are upregulated in WOX9-induced lines, but downregulated in STF/LAM1-induced lines (Figure 3, B and C; Supplemental Figures S3B and S8), suggesting that these CKX genes are direct targets of STF/LAM1 repression and WOX9 activation in both M. truncatula and N. sylvestris.

Figure 3.

Figure 3

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NsCKX3 is the common target of LAM1 and NsWOX9 transcriptional regulation. A, Relative transcript levels of NsCKX genes in the top first and second youngest leaves in wild-type, the lam1 mutant, and STFpro:MtWOX9 T3 transgenic plants in N. sylvestris. Data are shown as means ± standard deviation (sd) of three biological replicates of pooled samples from five different plants grown simultaneously. Individual replicates are shown as gray circles. B and C, Relative transcript levels of NsCKX3 after DEX induction in the top first and second leaves from two independent 35S:YGR-LAM1 (B) and 35S:YGR-NsWOX9 (C) T2 transgenic plants. Data are shown as means ± sd of three technical replicates of two independent transformants. D, Schematic diagram of the effector and reporter constructs used in dual luciferase assays. E, Dual luciferase assays showing the transcriptional activities of LAM1 and NsWOX9 on the NsCKX3 promoter in Arabidopsis protoplasts. Data are means ± sd of three technical replicates from pooled protoplasts divided into three samples for transient transfection. Asterisks indicate significant differences (*P < 0.05, **P < 0.01, Student’s t test).

NsCKX3 is directly regulated by LAM1 and NsWOX9 in N. sylvestris

To confirm that NsCKX3 is indeed oppositely regulated by LAM1 and NsWOX9, we performed a dual LUC assay in Arabidopsis protoplasts using a dual LUC assay system. We placed the firefly LUC reporter gene under the control of an ∼2 kb promoter region upstream of the translational start codon of NsCKX3, while Renilla (REN) LUC was driven by the cauliflower mosaic virus 35S promoter for normalization. All effector constructs expressing LAM1, NsWOX9, or GFP control were driven by the 35S promoter (Figure 3D). We co-transformed combinations of effector and reporter plasmids into Arabidopsis protoplasts, and measured luminescence. We observed that relative LUC activity is significantly downregulated in the presence of the LAM1 effector protein but upregulated in the presence of the NsWOX9 effector protein, compared to the GFP effector control (Figure 3E), indicating that the NsCKX3 promoter is repressed by LAM1 but activated by NsWOX9. Because LAM1 and NsWOX9 belong to the WOX family with a conserved DNA binding HD, we hypothesized that both LAM1 and NsWOX9 might recognize the same cis-elements in the NsCKX3 promoter. To test this hypothesis, we performed an electrophoretic mobility shift assay (EMSA) using three putative STF/LAM1 binding regions (P1, P2, and P3) in the proximal promoter region of NsCKX3 (Figure 4A) and recombinant His-TF-LAM1 and His-TF-NsWOX9. We established that both LAM1 and NsWOX9 can bind strongly to all three regions; in addition, binding was competed by excess unlabeled specific probes (Figure 4, B and C). To confirm that LAM1 and NsWOX9 can indeed recognize and bind to the NsCKX3 promoter in vivo, we performed chromatin immunoprecipitation (ChIP) assays, followed by qPCR, which revealed that all three promoter fragments are enriched in the ChIP samples prepared with anti-Myc antibody from 35S:Myc-NsWOX9 N. sylvestris leaves (Figure 4, D and F), and that two of the fragments, P1 and P3, are enriched in 35S:Myc-LAM1 samples (Figure 4, E and G). In contrast, we detected no enrichment using primers for the NsCKX3 coding region or for the UBIQUITIN gene NsUBI2, indicating that the enrichments are specific. Taken together, these results indicate that LAM1 and NsWOX9 not only directly bind to the NsCKX3 promoter in vitro and in vivo, but also specifically bind to the same cis-elements.

Figure 4.

Figure 4

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Both NsWOX9 and LAM1 directly bind to the NsCKX3 promoter. A, Schematic diagram of the NsCKX3 proximal promoter region, highlighting the three putative WOX-binding sites (P1–P3). B and C, EMSAs showing the binding of NsWOX9 (B) or LAM1 (C) HD to the three putative binding sites of the NsCKX3 promoter. Competition assays to the biotin-labeled probes were performed by adding 100- and 200-fold molar excess of unlabeled probe. Arrows indicate the shifted bands. D and E, Transcript levels of NsWOX9 (D) and LAM1 (E) in leaves co-infiltrated with 35S:Myc-NsWOX9 and 35S:Myc-LAM1 constructs, by RT-PCR. NsWOX9 and LAM1 were amplified with 35 cycles; NsUBI2 was amplified with 25 cycles. F–G, ChIP assays showing the enrichment of NsWOX9 (F) or LAM1 (G) at the indicated regions on the NsCKX3 promoter. Data are means ± sd of three biological replicates from pooled infiltrated leaves divided into three samples for incubation with c-Myc antibodies after total nucleoprotein extraction. Asterisks indicate significant differences (*P < 0.05, **P < 0.01, Student’s t test).

To provide further evidence that LAM1 and NsWOX9 can occupy the same cis-elements, thereby affecting the transcript levels of target genes, we conducted a dual LUC assay by expressing LAM1 and NsWOX9 as effectors together with the NsCKX3pro:LUC reporter construct. In the presence of LAM1 or NsWOX9 as effector, we measured lower or higher relative LUC activity from the NsCKX3 promoter, respectively, compared to the GFP control; however, the expression of both effectors largely neutralized this change, resulting in relative LUC activity comparable to that measured with the GFP control (Figure 5). To evaluate whether this neutralization of activity was caused by the repression and activation activities only or by competition to the NsCKX3 binding sites as well, we fused GFP to a truncated LAM1(del) in which the C-terminal domain of LAM1 that harbors the WUS-box and STF-box repressor domains was removed, and used the resulting GFP-LAM1(del) construct as effector. GFP-LAM1(del) exhibited no significant repressive effect on the NsCKX3pro:LUC reporter compared to the GFP control, in agreement with the removal of the repressor domain. However, when NsWOX9 and NsLAM1(del) were co-transfected, the accumulation of NsLAM1(del) partially compromised the activation activity of NsWOX9, as shown by reduced relative LUC activity compared to the NsWOX9 effector alone (Figure 5). This result indicates that LAM1 and NsWOX9 indeed physically compete for the same cis-elements in the NsCKX3 promoter via their respective HDs.

Figure 5.

Figure 5

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Dual luciferase assay showing the activity of GFP-NsWOX9 effector protein on the NsCKX3 promoter in the presence of LAM1 and LAM1(del) fusions. A, Schematic diagrams of the reporter and effector constructs used in the dual luciferase assays. LAM1(del), truncated LAM1 lacking the C-terminal repressor domains (WUS-box and STF-box, ΔC). B, Transcriptional activities of NsWOX9 toward the NsCKX3 promoter in the presence of LAM1 or its truncated fragment LAM1(del) in Arabidopsis transfected protoplasts. Data are means ± sd of three independent experiments with three technical replicates from pooled protoplasts divided into three samples for transient transfection. Different lowercase letters indicate significant differences (P < 0.05), as determined by a one-way ANOVA using Tukey’s honestly significant difference test as post hoc test.

Silencing of CKX3 partially rescues blade outgrowth in lam1 and stf mutants

To investigate the role of NsCKX3 in leaf blade development, we generated NsCKX3 overexpression and RNAi lines in N. sylvestris. Out of a total of 38 independent overexpression lines, 35 lines showed narrower and slightly shorter leaves, while nine out of 16 RNAi lines exhibited wider leaf blades without significant alteration in blade length, relative to nontransgenic plants (Figure 6, A–E), suggesting that NsCKX3 transcript levels are an important regulator of leaf blade expansion. Cellular imaging analysis revealed that epidermal cells are smaller in NsCKX3 RNAi lines but bigger in NsCKX3 overexpression lines compared to the wild-type (Figure 6, F–H), indicating that NsCKX3 reduces cell proliferation through promotion of cell expansion/differentiation. To confirm the genetic relationship between NsCKX3, LAM1, and WOX9, we also transformed the NsCKX3 RNAi construct into the lam1 mutant and MtWOX9 overexpression lines. Knockdown of NsCKX3 in the lam1 mutant background resulted in the partial rescue of the narrow leaf phenotype characteristic of the lam1 mutant, especially at very early stages of development (Figure 7, A–C). At later developmental stages, we noticed that the leaves become more branched rather than expanded in lam1 NsCKX3 RNAi lines (Figure 7, D–F), which was reminiscent of lam1 mutants treated with cytokinins (Tadege et al., 2011) and of lam1 mutant plants silenced for NsWOX9 (Wolabu et al., 2021). Similarly, we observed a partial restoration of blade outgrowth in the stf Mtckx3a Mtckx3b triple mutant of M. truncatula compared to the stf single mutant (Figure 7, G–J), while the Mtckx3a Mtckx3b double mutant showed no significant difference in either blade length or width relative to the wild-type R108 (Supplemental Figure S9). However, the epidermal cells of Mtckx3a Mtckx3b double mutant appeared slightly smaller than those of R108, but larger than stf mutant cells (Supplemental Figure S10), showing effects at the cellular level even though this difference may not be large enough to translate into a wider leaf blade in the double mutant. Silencing of NsCKX3 in N. sylvestris lines harboring the STFpro:MtWOX9 transgene and displaying mild phenotypes also almost fully rescued the abnormal leaf phenotypes (Supplemental Figure S11). These results suggest that the upregulation of NsCKX3 transcript levels partially accounts for the lam1 mutant phenotype and the effects of NsWOX9 overexpression in the wild-type and lam1 mutant backgrounds.

Figure 6.

Figure 6

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NsCKX3 RNAi and NsCKX3 overexpression alter leaf size in N. sylvestris. A–C, Phenotypes of 8-week-old wild-type N. sylvestris (A), a NsCKX3-RNAi T1 transgenic plant (B) and a 35S:NsCKX3 T1 transgenic plant (C). Scale bars = 10 cm. D and E, Leaf width (D) and length (E) in wild-type N. sylvestris, NsCKX3-RNAi and 35S:NsCKX3 plants. At least five second leaves from independent plants were measured using Image J software. Asterisks indicate significant differences (*P < 0.05, **P < 0.01, Student’s t test). F–H, Outlines of abaxial epidermal cells from the second leaflet middle area of wild-type N. sylvestris (F), NsCKX3-RNAi (G), and 35S:NsCKX3 plants (H). Scale bars = 50 µm.

Figure 7.

Figure 7

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Silencing of CKX3 partially rescues the leaf blade phenotypes of lam1 and stf mutants. A, Phenotype of NsCKX3-RNAi in the lam1 mutant background during early development. The arrows indicate the partially rescued leaf phenotype of positive transgenic plants in tissue culture. B and C, Phenotypes of a representative lam1 (B) or T0lam1 NsCKX3-RNAi (C) plant four weeks after transfer to soil. D and E, Phenotype of two independent lam1 NsCKX3-RNAi lines two months after transfer to soil. F, Close up of the leaf blade phenotype shown in (E) compared to untransformed lam1. G and H, Phenotype of 6-week-old stf single mutant (G) and stf Mtckx3a Mtckx3b (H) triple mutant plants. I, Close up of the leaf blade from the stf and stf Mtckx3a Mtckx3b mutants shown in (G) and (H). The arrows indicate the terminal leaflet (TL) used for width and length measurements in (J) and (K). J and K, Leaf width (J) and length (K) in stf and stf Mtckx3a Mtckx3b mutants. At least 10 totally opened leaves from independent plants were photographed, and blade width and length were measured using ImageJ software. Asterisk indicates significant difference (**P < 0.01, Student’s t test). Scale bars = 0.5 cm in (B, C, F, I, and J), and 2 cm in (A, D, E, G, and H).

Overexpression of NsCKX3 leads to narrower and shorter leaves in the lam1 mutant and STFpro:NsWOX9 ectopic lines

To further evaluate the consequences of NsCKX3 overexpression, we expressed NsCKX3 driven by the 35S promoter in the lam1 mutant and STFpro:MtWOX9 lines. We determined that overexpressing NsCKX3 in the lam1 mutant affects both the leaf length and width to a similar extent as the ectopic expression of MtWOX9 (driven by the STF promoter) in lam1, with significantly narrower and shorter leaf blades than the lam1 mutant, with higher expressing lines showing more severe phenotypes (Figure 8, A–C; Supplemental Figure S12). Likewise, 35S:NsCKX3 overexpression in the STFpro:MtWOX9 lines exhibited both smaller blade width and length compared to STPpro:MtWOX9 lines (Figure 8, D–G). These data collectively suggest that NsCKX3 expression is one of the factors that contribute to the phenotypes seen in the lam1 mutant and MtWOX9 ectopic expression lines, and that repression of NsCKX3 by LAM1 is required for proper leaf blade outgrowth in N. sylvestris.

Figure 8.

Figure 8

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Overexpression of NsCKX3 exacerbates the leaf phenotypes of the lam1 mutant and STFpro:MtWOX9 lines in N. sylvestris. A, Phenotypes of three independent 10-week-old NsCKX3 overexpression lines in the lam1 background, with lam1 35S:GFP as control. B and C, Leaf width (B) and length (C) of 10-week-old lam1 35S:NsCKX3 lines, as measured using ImageJ software. Data are means ± sd of 10 leaves, with individual leaves shown as gray circles. D, Phenotype of 5-week-old wild-type N. sylvestris. E, Phenotype of 5-week-old STFpro:MtWOX9 plants. F, Phenotype of a 5-week-old 35S:NsCKX3 STFpro:MtWOX9 plant. G, Leaf width and length of 5-week-old STFpro:MtWOX9 and 35S:NsCKX3 STFpro:MtWOX9 lines, as measured using ImageJ software. Data are means ± sd of five leaf measurements. Asterisks indicate significant differences (*P < 0.05, **P < 0.01, Student’s t test). Scale bars = 2 cm in A, 5 cm in (D–F).

Endogenous active cytokinin levels are reduced in the lam1 mutant and STFpro:NsWOX9 ectopic lines

Cytokinin oxidases/dehydrogenases catalyze the irreversible degradation of the cytokinin isopentenyladenine (iP), zeatin, and their ribosides (Schmulling et al., 2003; Zalabak et al., 2014). Since NsCKX3 is a common target repressed by LAM1 and activated by NsWOX9 in N. Sylvestris, we hypothesized that LAM1 and NsWOX9 may modulate local active cytokinin levels during leaf blade development by controlling the abundance of NsCKX3 transcripts. To test this hypothesis, we measured the main active cytokinins iP, trans-zeatin (tZ), and their ribosides (iPR, tZR, and cZR) in the youngest top two leaves of 6-week-old lam1 mutant and STFpro:MtWOX9 and 35S:NsCKX3 transgenic plants, using wild-type leaves as controls. We observed that iP, iPR, and tZ levels are significantly lower in the lam1 mutant compared to the wild-type, while tZR and cZR levels were unchanged. Similarly, iPR, tZ, and cZR were also distinctly reduced in STFpro:MtWOX9 lines, with the most dramatic decrease among all five cytokinins tested being detected in the NsCKX3 overexpressing lines (Figure 9). These results indicate that the NsCKX3 enzyme indeed functions in degrading active cytokinins in N. sylvestris, and suggests that the antagonistic regulation of its transcript abundance by LAM1 and NsWOX9 may control active cytokinin homeostasis required for cell proliferation and/or differentiation during leaf blade morphogenesis and maturation.

Figure 9.

Figure 9

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Active cytokinin levels are lower in lam1, STFpro:MtWOX9 and 35S:NsCKX3 lines in N. sylvestris. Endogenous cytokinin contents in the top two youngest leaves of 6-week-old plants. iPR, isopentenyladenine riboside; tZR, trans-zeatin riboside; cZR, cis-zeatin riboside. Data are means ± sd of three independent experiments from pooled samples of 20–25 different plants. Asterisks indicate significant differences (*P < 0.05, **P < 0.01, Student’s t test). FW, fresh weight.

LAM1 physically interacts with NsWOX9 to regulate NsCKX3 expression

The previous results demonstrated that NsCKX3 is not only the common target of LAM1 and NsWOX9, but also that both LAM1 and NsWOX9 can recognize and bind to the same cis-elements in the proximal NsCKX3 promoter (Figures 4 and 5), raising the possibility that these two proteins may physically interact to compete for the same binding sites. To test this possibility, we performed a yeast two-hybrid (Y2H) assay using full-length LAM1, as well as truncated LAM1(del) as baits, and full-length NsWOX9 as a prey. We determined that NsWOX9-GAL4 activation domain (AD) (a fusion between NsWOX9 and the AD of yeast GAL4) interacts strongly with LAM1-GAL4 BD and LAM1(del)-GAL4 binding domain (BD) (fusions to the DNA-BD of GAL4), as demonstrated by normal growth of yeast colonies on stringent quadruple dropout (QDO) medium and by the blue color of the colonies due to the presence of X-α-gal (Figure 10A). To confirm the interaction between LAM1 and NsWOX9 in living plant cells, we conducted bimolecular fluorescence complementation (BiFC) assays using the split YFP system. To this end, we fused full-length LAM1 and truncated LAM1(del) to the C-terminal half of YFP (YC), while full-length NsWOX9 was fused to the N-terminal half of YFP (YN). We introduced all resulting encoding constructs into Agrobacterium tumefaciens for Agrobacterium-mediated transient co-infiltration of Nicotiana benthamiana leaves, before looking for YFP fluorescence 36–48 h later by confocal microscopy. We established that both LAM1 and LAM1(del) physically interact with NsWOX9, as evidenced by yellow fluorescence, while negative controls showed no signals in plant cells (Figure 10B; Supplemental Figure S13). We further confirmed this interaction in vivo by co-immunoprecipitation (Co-IP) assays. Indeed, Myc-tagged NsWOX9 pulled down both LAM1 and LAM1(del), which carried a GFP tag (Figure 10C), validating the interaction between NsWOX9 and LAM1, and that the C-terminal-repressive domain of LAM1 is not required for this interaction. Taken together, our results demonstrate that repressor and activator WOX transcription factors antagonistically regulate a common target that modulates active cytokinin levels in leaves, thereby maintaining cytokinin homeostasis for cell proliferation and differentiation during leaf blade development. We propose that this may be a general mechanism by which evolutionarily distinct groups of WOX transcriptional regulators work together to control plant development and morphological diversity in flowering plants.

Figure 10.

Figure 10

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NsWOX9 physically interacts with LAM1. A, Protein–protein interactions between NsWOX9 and LAM1 by Y2H assay. DDO, double dropout medium (synthetic defined [SD] medium–Trp–Leu); (SD–Ade–His–Leu–Trp). B, BiFC assays showing the interaction of NsWOX9 and LAM1 in N. benthamiana leaf cells. Nuclear protein AHL22 (AT-HOOK MOTIF NUCLEAR-LOCALIZED PROTEIN 22) was used as a nuclear localization marker. Scale bars = 50 µm. C, Co-IP assay showing the physical interaction of NsWOX9 with LAM1 and LAM1(del) in total protein extracts from infiltrated leaves.

Discussion

Transcriptional activation and repression are fundamental mechanisms through which gene regulation and ultimately cellular life processes are controlled by transcription factors. The WOX family represents plant-specific homeobox transcription factors that are involved in the regulation of several key developmental programs, from embryo patterning to meristem maintenance and lateral organ development. Following the characterization of Arabidopsis WUS, the founding member of the WOX family, functioning in SAM maintenance (Laux et al., 1996; Mayer et al., 1998), diverse WOX functions have been described and important mechanistic insights are emerging (Leibfried et al., 2005), but detailed molecular mechanisms and relationships among WOX regulators are poorly understood. Here we described a potential mechanism for the antagonistic interaction of STF with MtWOX9 and that of LAM1 with NsWOX9 in the regulation of leaf blade development in the two unrelated species M. truncatula (Fabaceae) and N. sylvestris (Solanaceae), respectively. STF, LAM1, and WOX1 belong to the modern/WUS-clade of the WOX family, and function primarily via a transcriptional repression mechanism (van der Graaff et al., 2009; Lin et al., 2013). Medicagotruncatula STF and its homolog LAM1 in N. sylvestris are critically required for blade outgrowth in the mediolateral direction, and function by promoting cell proliferation (Tadege et al., 2011). However, this function is antagonized by the activity of MtWOX9 or NsWOX9 in their respective species, as evidenced by the narrower leaves of the stf and lam1 mutants with ectopic expression of MtWOX9 or NsWOX9 in the mutant background (Lin et al., 2013; Wolabu et al., 2021). WOX9 belongs to the intermediate/WOX9-clade (Haecker et al., 2004; van der Graaff et al., 2009), and exhibits a transcriptional activation activity in protoplast assays (Lin et al., 2013). We recently reported that MtWOX9 and NsWOX9 are required for leaf polarity patterning and proper leaf development, and promote cell differentiation (Wolabu et al., 2021). STF directly represses MtWOX9 expression to allow cell proliferation and prevent cell differentiation at the STF expression domain in the adaxial–abaxial juxtaposition, but ectopic WOX9 expression resulted in narrower and shorter stf and lam1 mutant leaves, and silencing of NsWOX9 partially rescued the lam1 mutant phenotype (Supplemental Figure S1; Wolabu et al., 2021). In this report we investigated the underlying mechanisms of this antagonistic activity.

We established that LAM1 directly binds to the NsCKX3 promoter and represses its transcription to promote active cytokinin levels, thereby activating cell proliferation (Figures 3, 4, and 9). In Arabidopsis, WOX1, PRS, and WOX5 were shown to redundantly control auxin biosynthesis along the leaf margin through regional control of YUCCA gene expression to control leaf shape and form (Zhang et al., 2020). Our results are consistent with previous reports of auxin and cytokinins acting as key players in the STF pathway, and of how the combined application of auxin and cytokinins to the shoot tip of the lam1 mutant partially rescues the mutant leaf phenotype (Tadege et al., 2011; Tadege and Mysore, 2011; Tadege, 2016). Activation of cytokinin signaling by STF was also implicated in transgenic rice and switchgrass (Panicum virgatum L.) that ectopically express STF (Wang et al., 2017). Consistent with this notion, Arabidopsis WUS promotes cytokinin activity in the shoot stem cell niche by repressing the expression of type-A ARR genes (Leibfried et al., 2005) to activate cell proliferation. Type-A ARR genes are negative regulators of cytokinin signaling (To et al., 2004). Thus, while the activation of cytokinin signaling by a WOX member is not surprising, doing so by repressing the expression of genes encoding cytokinin degrading enzymes provides a novel perspective in the control of local active cytokinin levels by WOX transcriptional regulators. Cytokinins promote cell proliferation by shortening cell cycle phases and delaying the onset of cell differentiation (Zhang et al., 2005; Holst et al., 2011; Skalak et al., 2019; Wu et al., 2021). Indeed, cell proliferation was shortened in transgenic Arabidopsis leaves that accelerate the degradation of cytokinins by CKX3 expression under the AINTEGUMENTA promoter, while cell expansion was accelerated, leading to smaller leaves with fewer cells (Holst et al., 2011). Although this mechanism may not be specific to blade lateral expansion, these results indicate that CKX3 could be deployed by specific transcription factors to control specific growth in lateral organs.

Notably, we determined that NsWOX9 also binds to the NsCKX3 promoter but, unlike LAM1, behaves as a transcriptional activator instead of a repressor (Figures 3–5), indicating that both LAM1 and NsWOX9 target NsCKX3 to oppositely regulate its transcript levels, uncovering the molecular basis for LAM1 and NsWOX9 antagonistic activity. Since CKX3 accelerates cell differentiation and reduces cell proliferation by lowering active cytokinin levels in transgenic Arabidopsis leaves (Holst et al., 2011), our observation of small leaves and accelerated cell differentiation in NsWOX9 overexpressing N. sylvestris lines (Wolabu et al., 2021) is consistent with the accelerated degradation of cytokinins via activation of NsCKX3 transcription by NsWOX9. Indeed, CKX3 transcript levels increased in lines ectopically expressing MtWOX9/NsWOX9 and decreased in WOX9-RNAi lines in both transgenic M. truncatula and N. sylvestris (Supplemental Figures S4 and S7). To provide a direct support for this interpretation, we manipulated the levels of CKX3 transcripts in the lam1 mutant background using genetic and transgenic approaches. We showed that silencing of NsCKX3 by RNAi in the lam1 background partially rescues the mutant leaf phenotype, especially at early stages of leaf development (Figure 7), and that silencing of NsCKX3 in the STFpro:MtWOX9 background nearly fully rescues the mild leaf phenotypes (Supplemental Figure S11). Similarly, generating the stfMtckx3a Mtckx3b triple mutant revealed a substantial rescue of the stf mutant leaf phenotype in M. truncatula by the loss of MtCKX3A and MtCKX3B function (Figure 7, G–K). Although leaves of the Mtckx3a Mtckx3b double mutant were of comparable size to those of the wild type (Supplemental Figure S9), the mutant epidermal cells were slightly smaller than those of the wild-type R108; in the partially rescued stf Mtckx3a Mtckx3b triple mutant, epidermal cells appeared shorter than the stf single mutant (Supplemental Figure S10), suggesting that MtCKX3A and MtCKX3B redundantly function in promoting cell expansion/differentiation. This notion is in agreement with the observation of Arabidopsis ckx mutants, as the ckx3 ckx5 double mutant displays more lateral organs and larger inflorescence meristems than the wild type (Bartrina et al., 2011). Moreover, overexpression of NsCKX3 in N. sylvestris resulted in narrower leaves, and exacerbated the leaf phenotypes in the lam1 mutant and ectopic WOX9 expression lines with additional defects in leaf length that were not observed in the original lam1 mutant (Figures 6 and 8). These experiments clearly indicate that the stf and lam1 mutant leaf phenotypes, as well as the phenotypes of WOX9 overexpression lines, can at least in part be attributed to an upregulation of NsCKX3/MtCKX3 transcript levels, suggesting that local cytokinin homeostasis is critical to balance cell proliferation and differentiation during leaf blade development in M. truncatula and N. sylvestris. MtCKX3 and NsCKX3 serve as central regulators of active cytokinin levels, and their activity is modulated by the antagonistic transcriptional control of their encoding genes by STF and MtWOX9 in M. truncatula, and by LAM1 and NsWOX9 in N. sylvestris. However, the phenotypic rescue of the stf and lam1 mutants by the Mtckx3a Mtckx3b double mutant in stf or by NsCKX3-RNAi in lam1 was not complete, suggesting that STF/LAM1 or the STF/MtWOX9 and LAM1/NsWOX9 modules also control additional targets or phytohormones to regulate blade expansion. For example, we showed previously that both the stf and lam1 mutant leaves contain significantly lower levels of free auxin compared to their respective wild types (Tadege et al., 2011), consistent with the recent report that Arabidopsis WOX1 controls auxin biosynthesis in the leaf margin (Zhang et al., 2020). Future experiments should focus on putting the remaining components together to fully rescue the lam1 mutant phenotypes.

It should be noted that both LAM1 and NsWOX9 directly bind to the NsCKX3 promoter to repress and activate its transcription, respectively, but importantly they bind to two of the same binding sites out of the three cis-elements tested in vivo (Figure 4). This result suggests that LAM1 and NsWOX9 compete for the same NsCKX3 promoter binding sites; this competition may be critical in allowing cell proliferation or cell differentiation to proceed depending on the need and stage of leaf development. The fact that LAM1 and NsWOX9 physically interact (Figure 10) also suggests that they may interact to physically compete for the binding sites. However, the upstream factors that regulate the relative abundance of the respective transcripts or other factors that help prioritize binding are unknown. In Arabidopsis, adaxially abundant MONOPTEROS (MP, also named AUXIN RESPONSE FACTOR 5 [ARF5]) directly activates WOX1 and PRS expression, while abaxially abundant ARF2, ARF3, and ARF4 suppress WOX1 and PRS transcription to specify and define the WOX1 and PRS expression pattern in the middle domain for leaf blade flattening (Guan et al., 2017). However, whether ARF2-5 homologs can regulate the expression of STF and LAM1 in M. truncatula and/or N. sylvestris remains to be shown. More importantly, whether these factors similarly or antagonistically control WOX9 expression in Arabidopsis has not been demonstrated, although it is tempting to speculate that ARF2, ARF3, and ARF4 may activate WOX9 expression, since MtWOX9 is abaxially expressed in M. truncatula (Wolabu et al., 2021). It is, therefore, plausible that auxin signaling prepatterns and modulates the LAM1 and NsWOX9 competition for the NsCKX3-binding site, and thus local active cytokinin levels, to regulate cell proliferation and differentiation during leaf blade lateral outgrowth. Despite the existence of numerous reports describing the crosstalk between auxin and cytokinins in several plant developmental programs (Dello loio et al., 2008; Bartrina et al., 2011; Su et al., 2011; Zhang et al., 2011; Della Rovere et al., 2013; Zhang et al., 2013; Chandler and Werr, 2015; Hussain et al., 2021; Wu et al., 2021), this hypothesis of modulation of LAM1 and NsWOX9 activity via auxin signaling to maintain cell proliferation and differentiation homeostasis by cytokinin signaling in leaf development remains to be experimentally tested. Irrespective of how the competition between LAM1 and NsWOX9 for the NsCKX3 promoter is modulated or how the relative abundance of their encoding transcript levels is controlled, our data clearly demonstrate that the antagonistic regulation of active cytokinin levels through controlling cytokinin degradation by modern clade and intermediate clade WOX members orchestrate the cell proliferation and differentiation homeostasis required for leaf blade development. This mechanistic insight sheds new light on the role of coordinated WOX activity in regulating plant morphology, growth, and development.

Materials and methods

Plant materials and growth conditions

All plant materials used in this study were grown in a greenhouse at 24°C (day) and 20°C (night) under a 16-h light/8-h dark photoperiod, 60%–70% relative humidity and a light intensity of 180 µmol·m−2 s−1. Light was provided by white-light tubes (400–700 nm). The M. truncatula Mtckx3a (NF11391), and Mtckx3b (NF5949) mutants were donated by Prof. Hao Lin. The stf Mtckx3a Mtckx3b triple mutant was obtained by crossing a stf/STF plant to the Mtckx3a Mtckx3b double mutant. MtWOX9 overexpression in the stf background was obtained by crossing a stf/STF plant to the MtWOX9 overexpression line and selection of the desired combination in the progeny.

Plasmid construction and plant transformation

For overexpression lines, the full-length coding sequences of MtWOX9 and STF were cloned from M. truncatula ecotype R108, and the full-length coding sequences of NsWOX9, LAM1, and NsCKX3 were cloned from N. sylvestris. All coding sequences were introduced into the Gateway entry vector pDONR207 via BP reaction (Invitrogen, Gateway BP Clonase) and then recombined into the destination vector pMDC32 by LR reaction as indicated in the user manual (Invitrogen, Gateway LR Clonase). For the DEX-inducible construct 35S:YGR-NsWOX9, YFP-GR (YGR) and NsWOX9 were cloned separately, with 18 bp of overlapping sequence between the 3′-end of YGR and the 5′-end NsWOX9 to obtain the YGR-NsWOX9 fragment flanked with attB primers, and then recombined into pMDC32 by LR reaction to obtain 35S:YGR-NsWOX9. The same procedure was followed to obtain 35S:YGR-LAM1, 35S:YGR-MtWOX9, and 35S:YGR-STF. The resulting constructs were introduced into Agrobacterium (A.tumefaciens) by freeze–thaw transformation. The Agrobacterium strains AGL1 and GV2260 were used for M. truncatula and M. sylvestris transformation, respectively, as described (Tadege et al., 2011).

For M. truncatula transformed plants, seed coats were rubbed on the sandpaper and then geminated in water containing 50-µM hygromycin before being transferred to soil. Seeds of N. sylvestris transformants were surface sterilized and germinated on half-strength Murashige and Skoog medium containing 50-µM hygromycin for overexpression lines and 100-µM kanamycin sulfate for RNAi lines. DNA was extracted from primary transformants and confirmed by PCR, before at least three independent lines were tested by RT-qPCR for their transcript levels. All primers used here are listed in Supplemental Table S1.

RNA extraction and RT-qPCR

Total RNA was extracted from the leaves indicated using TRIzol reagent (Invitrogen, Waltham, MA, USA, 15596018), 4 µg of total RNA was used as template for cDNA synthesis with Superscript III reverse transcriptase (Invitrogen, 18080-044) following the manufacturer’s instructions. qPCR was performed using a StrepOne Plus system (Applied Biosystems, Waltham, MA, USA) with PowerUp SYBR Green (A25741, Thermo Fisher, Waltham. MA, USA) and qTOWER3 G (Analytik Jena, Jena, Germany) with TransStart Green qPCR SuperMix (Transgen, AQ101-01). Transcript levels were normalized to the control MtACTIN (Medtr3g095530) in M. truncatula and NsUBC2 (XM_009778452.1) in N. sylvestris. Young folded leaves (at the P6 stage) in M. truncatula and the top first and second leaves in N. sylvestris were harvested for RT-qPCR analysis. The analyses were performed as three replicates from independent pooled samples of 5–8 different plants grown simultaneously. For both DEX and CHX treatments, RT-qPCR was conducted on three replicates of independent pooled samples from eight different plants from two independent transgenic lines. Student’s t tests were used to determine the significance of differences in transcript levels. All primers used are listed in Supplemental Table S1.

RNA-seq analysis and heatmap plotting

For RNA-seq analysis, 4-week-old independent transformants of 35S:YGR-MtWOX9 were sprayed with CHX (10 µM) or CHX and DEX (10 µM each) diluted in 0.015% (v/v) Silwet-77. Folded leaves (P5 stage) were harvested 3 h after treatment and total RNA was extracted with TRIzol reagent (Invitrogen). RNA integrity was evaluated and libraries were constructed and sequenced by Novogene.

For the identification of MtWOX9 downstream genes, high-quality sequenced reads from RNA-seq libraries were mapped to the reference genome (Mt4.0, 2014) using HISAT2. Differential gene expression analysis was done using DESeq (Anders and Huber, 2010), and P-values were estimated by the negative binomial distribution model. For selecting differentially expressed genes, the threshold was set as P < 0.01 and a |Log2(Fold-change)| > 2. The heatmap and volcano plots inferring the expression patterns and overall distribution of the DEGs were generated using heatmap.2 and ggplot functions in R, respectively.

Measurements of cytokinin contents

Approximately 1 g of fresh young leaves from 6-week-old plants were harvested and immediately frozen in liquid nitrogen. Extraction and determination of endogenous cytokinin contents were performed by using a polymer monolith microextraction/hydrophilic interaction chromatography/electrospray ionization–tandem mass spectrometry method by Wuhan Greensward Creation Technology Company (Liu et al., 2010). The analyses were performed on three independent pooled samples from 20 to 25 different plants in total. Student’s t test was used to determine the significance of differences.

Dual LUC assay

For effector plasmids, the coding sequences of LAM1 or LAM1(del) and NsWOX9 were fused to that of GFP as described (Wang et al., 2017) and cloned into the entry vector pDONR207, before being recombined into p2GW7 by LR reaction. For the reporter plasmid, a 2-kb promoter fragment from NsCKX3 was cloned into the destination vector pGreenII0800-LUC at the restriction sites PstI and SacI to obtain NsCKX3pro:LUC as reporter construct. A transient transcriptional activity assay was performed via the transient transfection of protoplasts using dual LUC according to a previously described method (Wang et al., 2017) using the firefly/REN LUC system (Promega E1910, Madison, WI, USA). Each 5 µg of effector and reporter were co-transfected into 100 µL of Arabidopsis protoplasts using the PEG-calcium transfection method and incubated in the dark for 16–18 h at room temperature. The relative LUC/REN activities were measured with a dual-LUC reporter assay system (Promega) on a Promega GloMax Multi JR instrument. All transfections were performed in triplicate to calculate the LUC and REN LUC activities. The data are reported as the mean of three technical repeats and a Student’s t test was used to assess for significance differences. The experiments were performed 3 times.

EMSA

Fragments of the LAM1 and NsWOX9 coding sequences encoding the N-terminal and HD regions, respectively, were cloned into the pCOLD/TF vector (Takara, Kusatsu, Japan) by BamHI and SalI digestion for His-tag fusion constructs and were transformed into Escherichiacoli strain BL21 (DE3) for protein production. His fusion proteins were purified using Profinity IMAC Ni-Charged Resin (BIO-RAD, Hercules, CA, USA) according to the manufacturer’s protocol and quantified with a Bio-Rad protein assay reagent. For EMSA, recombinant His-TF-LAM1 and His-TF-NsWOX9 were eluted from the Ni resin and desalted using Millipore Amicon Ultra for exchanging phosphate buffered saline with 10% (v/v) glycerol. For DNA probes, three pairs of DNA fragments within the 2-kb region upstream of the translation start codon were synthesized and labeled with biotin using a DNA 3′-End Biotin labeling Kit (Thermo; 89818), and then denatured and annealed as double-stranded probes for reaction. The reactions were incubated at room temperature for 20 min and then resolved on a 6% (w/v) DNA retardation gel by electrophoresis at 100 V for 1–2 h and transferred to a nylon membrane. Biotin signals were detected using the LightShift Chemiluminescent EMSA Kit (Thermo; 20148) as suggested by the manufacturer.

ChIP assay

The ChIP assays were performed as described previously (Saleh et al., 2008; Wang et al., 2017). Leaves co-infiltrated with Agrobacterium (strain GV2260) cultures harboring 35S:myc-NsWOX9 and 35S:myc-LAM1 were harvested after 48 h in 1% (w/v) formaldehyde in crosslinking buffer (0.4-M sucrose, 10-mM Tris–HCl pH 8.0, 1 cocktail in 50-mL buffer). Crosslinking was stopped by the addition of 2-M glycine (final concentration of 100 mM). Leaves were washed and then ground to a fine powder in liquid nitrogen, the nuclear pellet was isolated with nuclei lysis buffer (50-mM Tris–HCl pH 8.0, 1% [v/v] Triton X-100, 167-mM NaCl, 1-mM EDTA). The DNA was then sheared into ∼500-bp fragments on ice by sonication. The sheared chromatin was precleared on salmon sperm-sheared DNA/protein A agarose beads. Precleared chromatin (50 μL) was put aside as Input, while the remaining supernatant was divided into two equal volumes, and incubated with 5 µL of anti-Myc antibody (9E10, Invitrogen). After overnight incubation at 4°C, protein A agarose beads (40 µL) were added to each sample and incubated at 4°C for 2 h. Beads were then washed with successive low-salt, high-salt, LiCl, Tris–HCl EDTA pH 8.0 (TE) buffers, and resuspended in elution buffer, incubated at 65°C for 4 h to reverse the crosslink. DNA was purified and diluted 10-fold, and 3 µL of DNA was used as a template for each qPCR amplification. Two independent experiments were performed.

Y2H

NsWOX9-AD, LAM1-BD, and LAM1(del)-BD clones for Y2H assays were cloned into the Gateway (Invitrogen) version of pGBKT7-GW and pGADT7-GW vectors. Sets of constructs were co-transformed into the Y2H gold yeast strain (Clontech). Protein interaction tests were assessed on QDO medium lacking histidine, tryptophan, leucine, and adenine in the presence of 4-mg mL−1 X-α-gal.

BiFC assays

BiFC assays were conducted using the split YFP system as described (Lu et al., 2010). Briefly, the NsWOX9 coding sequence was cloned into pEarleygate201-YN while those of LAM1 and LAM1(del) were cloned into pEarleygate202-YC by LR reaction. The NsWOX9-YN and LAM1-YC or LAM1(del)-YC were introduced into Agrobacterium strain GV2260 and co-infiltrated as appropriate pairs, together with the P19 suppressor, into 4-week-old N. benthamiana leaves. YFP signal was observed 48–60 h after infiltration by Nikon’s A1 confocal laser scanning microscopy (excitation and emission wavelength at 488 and 510 nm for YFP, 543 and 610 nm for RFP [mCherry]).

Co-IP

The leaves of 4-week-old N. benthamiana plants were co-transfected with Agrobacterium cell suspensions harboring the constructs pGWB521-NsWOX9 and pEarleygate104-LAM1 or pEarleygate104-LAM1(del). The leaves were harvested 3 days after infiltration. About 0.5 g of leaves were ground and homogenized in 2-mL protein lysis buffer (50 mM Tris–HCl pH 8.0, 150-mM NaCl, 10% [v/v] glycerol, 2-mM EDTA, 1 cocktail in 50-mL buffer) to extract total proteins. An aliquot of 50 µL of each protein extract was kept as an input; 2 mL of protein extract was incubated with 5-µL anti-GFP antibody (Abcam, Cambridge, UK, ab290) at 4°C overnight, and then 20-µL protein A agarose beads were added and incubated for 2 h at 4°C. After incubation, the beads were washed and boiled in 100-µL SDS loading buffer and subjected to 10% (w/v) SDS-polyacrylamide gel electrophoresis (SDS–PAGE), followed by immunoblotting. LAM1-GFP or LAM1(del)-GFP and NsWOX9-MYC were detected with anti-GFP (Abcam; ab290) and anti-Myc (Invitrogen; 9E10) antibodies (diluted at 1/10,000 for immunoblotting), respectively.

RNA in situ hybridization

The RNA in situ hybridization was performed as described previously (Corn et al., 1990; He et al., 2020). The probe was amplified from the vector pDONR207-MtWOX9-CDS using the specific primers F6167 and F6177 with a T7 promoter sequence in the reverse primer. The probe was then transcribed with T7 and SP6 RNA Polymerases and labeled using the DIG oligonucleotide 3′-end labeling kit. 3-week-old V-shoots were fixed in FAA fixative for 3 h, and then dehydrated and paraffin embedded using LEICA ASP200S and LEICA EG1150C/H. The wax blocks were cut into 8-µm sections using LEICA RM2235 and kept the slides overnight on 42°C heating plate (LEICA HI1210). The slides were hybridized with the digoxigenin-labeled probe and photographed using OLYMPUS BX63, sense probes were used as a negative control.

Sequence alignment and phylogenetic analysis

To obtain homologous sequences of CKX proteins, the full-length of the AtCKX1 protein sequence was used as a query forBLASTP against the A.s thaliana, M.truncatula, Oryza sativa databases in Phytozome, and the N.sylvestris database at the National Center for Biotechnology Information (NCBI). The resulting 35 protein sequences were aligned by the MAFFT method from (Madeira et al., 2022), and the unrooted phylogenetic tree was constructed by MEGA version 11 software (Tamura et al., 2021) using the neighbor-joining method (Saitou and Nei, 1987). The Poisson correction method was used to compute the evolutionary distances. To estimate the reliability of the constructed phylogenetic tree branches, the bootstrap method with 1,000 replicates was used, and the optimal tree is shown.

Statistical analysis

Error bars in RT-qPCR and dual LUC assay figures show the standard deviation of three biological or technical replicates, as indicated in the legends. Most of the pairwise comparisons between the means were performed using a two-sided Student’s t test, using GraphPad Prism version 9 software (Supplemental Data set 1).

Accession numbers

Sequence data from this article can be found in GenBank under the following accession numbers: STF (Medtr8g107210), MtWOX9 (Medtr2g015000), MtCKX3A (Medtr2g039410), MtCKX3B (Medtr4g126150), MACTIN2 (Medtr3g095530), NsUBC2 (XM_009778452.1), LAM1 (XM_036643044.1), NsWOX9 (XM_009794999.1), NsCKX1 (LOC_104218779), NsCKX1L (LOC_104231578), NsCKX3 (LOC_104240791), NsCKX3L1 (LOC_104245285), NsCKX3L2 (LOC_104237920), NsCKX5 (LOC_104213285), NsCKX6 (LOC_104232618), and NsCKX7 (LOC_104230796). Raw sequencing data have been deposited at the Gene Expression Omnibus at NCBI under accession number PRJNA841022, which can be downloaded at http://www.ncbi.nlm.nih.gov/sra/PRJNA841022.

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Overexpressing MtWOX9 exacerbates the stf mutant phenotypes in M. truncatula.

Supplemental Figure S2. Phenotypes of the 35S:YGR-MtWOX9 induced line.

Supplemental Figure S3. Volcano plot for the transcriptomes of 35S:YGR-MtWOX9 lines induced with DEX and CHX treatment compared to CHX control.

Supplemental Figure S4. MtCKX3 transcript levels decrease in MtWOX9 RNAi lines and increase in MtWOX9 overexpression lines of M. truncatula.

Supplemental Figure S5. MtCKX3 expression levels in stf mutant and MtWOX9 overexpressing stf mutant line of M. truncatula.

Supplemental Figure S6. Phylogenetic analysis of Cytokinin Oxidases in rice, Arabidopsis, M. truncatula, and N. sylvestris.

Supplemental Figure S7. NsCKX3 expression levels in STFpro:MtWOX9 transformed lines in N. sylvestris.

Supplemental Figure S8. MtCKX3 is directly repressed by STF in GR inducible lines in M. truncatula.

Supplemental Figure S9. The Mtckx3a Mtckx3b double mutant has no distinctive phenotype in M. truncatula.

Supplemental Figure S10. STF and MtCKX3 antagonistically regulate cell proliferation in M. truncatula.

Supplemental Figure S11. NsCKX3 RNAi nearly fully complements the mild leaf blade phenotypes of STFpro:MtWOX9 expressing lines in N. sylvestris.

Supplemental Figure S12. NsCKX3 expression levels in lam1 35S:NsCKX3 plants.

Supplemental Figure S13. Negative controls for physical interactions between NsWOX9 with LAM1 and LAM1(del) using BiFC assay.

Supplemental Table S1. Primers used in this study.

Supplemental Data Set 1. Summary of statistical analyses.

Supplemental File S1. Multiple protein sequence alignment for the phylogenetic tree shown in Supplemental Figure S6.

Supplemental File S2. Newick file format of the phylogenetic tree shown in Supplemental Figure S6.

Supplementary Material

koac188_Supplementary_Data
Click here for additional data file. (2.9MB, zip)

Acknowledgments

We thank Prof. Hao Lin and Prof. Lifang Niu (Biotechnology Research Institute, CAAS) for donating the Mtckx3a and Mtckx3b mutants, and we also thank Wuhan Greensword Creation Technology Co., Ltd for cytokinin measurements.

Funding

This project was supported in part by the National Science Foundation (NSF) grant IOS-1354422, Agriculture and Food Research Initiative Grant No. 2021-07493 from the USDA National Institute of Food and Agriculture, an Oklahoma Center for the advancement of Science and Technology (OCAST) grant PS21-006 to MT, and the National Natural Science Foundation of China (32000156) to HW.

Conflict of interest statement. None declared.

Contributor Information

Hui Wang, College of Grassland Science and Technology, China Agricultural University, Beijing 100193, China; Department of Plant and Soil Sciences, Institute for Agricultural Biosciences, Oklahoma State University, Ardmore, Oklahoma 73401, USA.

Xue Li, College of Grassland Science and Technology, China Agricultural University, Beijing 100193, China.

Tezera Wolabu, Department of Plant and Soil Sciences, Institute for Agricultural Biosciences, Oklahoma State University, Ardmore, Oklahoma 73401, USA.

Ziyao Wang, College of Grassland Science and Technology, China Agricultural University, Beijing 100193, China.

Ye Liu, Division of Life Sciences and Medicine, Hefei National Laboratory for Physical Sciences at the Microscale, CAS Center for Excellence in Molecular Plant Sciences, School of Life Sciences, University of Science and Technology of China, Hefei, China.

Dimiru Tadesse, Department of Plant and Soil Sciences, Institute for Agricultural Biosciences, Oklahoma State University, Ardmore, Oklahoma 73401, USA.

Naichong Chen, Department of Plant and Soil Sciences, Institute for Agricultural Biosciences, Oklahoma State University, Ardmore, Oklahoma 73401, USA.

Aijiao Xu, College of Grassland Science and Technology, China Agricultural University, Beijing 100193, China.

Xiaojing Bi, College of Grassland Science and Technology, China Agricultural University, Beijing 100193, China.

Yunwei Zhang, College of Grassland Science and Technology, China Agricultural University, Beijing 100193, China.

Jianghua Chen, CAS Key Laboratory of Topical Plant Resources and Sustainable Use, CAS Center for Excellence in Molecular Plant Sciences, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, Yunnan 650223, China.

Million Tadege, Department of Plant and Soil Sciences, Institute for Agricultural Biosciences, Oklahoma State University, Ardmore, Oklahoma 73401, USA.

 

H.W. and M.T. designed the research, H.W., X.L., T.W., Z.W., D.T., N.C., A.X., and Y.L. performed research, H.W., X.B., Y.Z., J.C., and M.T. analyzed the data, and H.W. and M.T. wrote the article.

The authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell) are: Hui Wang (huiwang211@cau.edu.cn) and Million Tadege (million.tadege@okstate.edu).

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Associated Data

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Supplementary Materials

koac188_Supplementary_Data
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