Calcium signaling contributes to xylem vessel cell differentiation via post-transcriptional regulation of VND7 downstream events

Abstract

Secondary cell walls (SCWs) accumulate in specific cell types of vascular plants, notably xylem vessel cells. Previous work has shown that calcium ions (Ca²⁺) participate in xylem vessel cell differentiation, but whether they function in SCW deposition remains unclear. In this study, we examined the role of Ca²⁺ in SCW deposition during xylem vessel cell differentiation using Arabidopsis thaliana suspension-cultured cells carrying the VND7-inducible system, in which VND7 activity can be post-translationally upregulated to induce transdifferentiation into protoxylem-type vessel cells. We observed that extracellular Ca²⁺ concentration was a crucial determinant of differentiation, although it did not have consistent effects on the transcription of VND7-downstream genes as a whole. Increasing the Ca²⁺ concentration reduced differentiation but the cells could generate the spiral patterning of SCWs. Exposure to a calcium-channel inhibitor partly restored differentiation but resulted in abnormal branched and net-like SCW patterning. These data suggest that Ca²⁺ signaling participates in xylem vessel cell differentiation via post-transcriptional regulation of VND7-downstream events, such as patterning of SCW deposition.

Introduction

Xylem vessels transport water and soluble minerals within vascular plants. Like other water-transporting cells in land plants, xylem vessel cells undergo programmed cell death (PCD) during differentiation; the vacuoles collapse as they digest nuclear and intracellular organelles (Bollhöner et al. 2012; Fukuda 2004; Turner et al. 2007). Before PCD is complete, xylem vessel cells deposit secondary cell walls (SCWs) between the primary cell walls and plasma membranes (Fukuda 2004; Nakano et al. 2015; Turner et al. 2007). SCWs are characterized by lignification, which provides high strength and water resistance to xylem vessel cells (Fukuda 2004; Nakano et al. 2015; Turner et al. 2007). Both PCD and SCW deposition are regulated by a specific group of NAC transcriptional factors, VASCULAR-RELATED NAC-DOMAIN (VND) family proteins, and their homologous VNS proteins (VND-, NST/SND- and SMB-related proteins) (Kubo et al. 2005; Nakano et al. 2015; Ohtani and Demura 2019; Ohtani et al. 2017; Yamaguchi et al. 2008). VND family proteins directly or indirectly regulate the genes involved in PCD and SCW formation, including those encoding proteases, cell wall biosynthetic enzymes, and SCW-related transcription factors (Ohashi-Ito et al. 2010; Yamaguchi et al. 2011; Zhong et al. 2010).

Calcium ions (Ca²⁺) are indispensable signaling molecules in eukaryotic cells and function broadly in plant growth and development (Hepler 2005). Pharmacological studies using a tracheary element induction system in Zinnia elegans mesophyll cells have demonstrated the importance of signaling mediated by Ca²⁺ and the Ca²⁺-binding protein calmodulin (CaM) in xylem vessel cell differentiation (Groover and Jones 1999; Iakimova and Woltering 2017; Kobayashi and Fukuda 1994; Roberts and Haigler 1990). Increased levels of CaM protein expression and membrane-bound Ca²⁺ are observed at early stages of tracheary element differentiation, and treatment with calcium-channel blockers and CaM inhibitors strongly inhibits tracheary element differentiation (Kobayashi and Fukuda 1994; Roberts and Haigler 1990), suggesting that xylem vessel cell differentiation depends on extracellular Ca²⁺. Treatment with inducers of Ca²⁺ release from intracellular stores also inhibits tracheary element differentiation (Roberts and Haigler 1990). Thus, both influx of extracellular Ca²⁺ and efflux of intracellular Ca²⁺ are crucial for xylem vessel cell differentiation (Iakimova and Woltering 2017). Groover and Jones (1999) showed that influx of extracellular Ca²⁺ is related to the induction of PCD: the influx of extracellular Ca²⁺ triggered by extracellular proteolysis induced vacuole collapse leading to DNA fragmentation (Bollhöner et al. 2012; Groover and Jones 1999). Additionally, the activation of phospholipase C by the influx of extracellular Ca²⁺ and subsequent generation of inositol triphosphates, important second messenger molecules, also have crucial functions in tracheary element differentiation (Barceló 1998; Zhang et al. 2002). Generation of H₂O₂/O₂⁻, which can induce lignification, requires metabolic pathways including CaM, inositol triphosphates, and protein phosphorylation (Barceló 1998). These observations in zinnia strongly suggest that Ca²⁺ signaling is essential in both PCD and SCW deposition during xylem vessel cell differentiation. However, the detailed function of Ca²⁺ signaling in SCW deposition, especially SCW patterning specific to xylem vessels, is still unclear.

In this study, we examined the function of Ca²⁺ signaling in SCW deposition in xylem vessel cell differentiation with Arabidopsis thaliana (Arabidopsis) suspension-cultured cells (T87 line) carrying the VND7-inducible system, in which protoxylem-type vessel cells can be induced by glucocorticoid application (Yamaguchi et al. 2010). We evaluated the effects of extracellular Ca²⁺ concentration on xylem vessel cell differentiation by varying the Ca²⁺ concentration of the culture medium and observed that extracellular Ca²⁺ concentration affected the efficiency of SCW deposition (i.e., the number of cells with SCWs) but did not consistently influence the upregulation of VND7-downstream genes. Treatment with voltage-gated calcium-channel inhibitors partly restored SCW deposition but affected SCW patterning, resulting in abnormal branched and net-like structures. These observations suggest that the active regulation of Ca²⁺ signaling is important for the post-transcriptional regulation of VND7-downstream gene expression and the regulation of SCW deposition.

Materials and methods

Plant material and growth conditions

Arabidopsis suspension-culture cell line T87 carrying the VND7 induction system (T87/35S::VND7-VP16-GR) (Roumeli et al. 2020; Yamaguchi et al. 2010) was used in this study. Cells were grown in 90 ml liquid medium (20 mg l⁻¹ 50× macronutrients [82.5 g l⁻¹ NH₄NO₃, 95 g l⁻¹ KNO₃, 8.5 g l⁻¹ KH₂PO₄, 0.31 g l⁻¹ H₃BO₃, 1.115 g l⁻¹ MnSO₄∙4H₂O, 0.43 g l⁻¹ ZnSO₄∙7H₂O, 41.5 mg l⁻¹ Kl, 12.5 mg l⁻¹ Na₂MoO₄∙2H₂O, 1.25 mg l⁻¹ CuSO₄∙5H₂O, 1.25 mg l⁻¹ CoCl₂∙6H₂O], 0.44 g l⁻¹ CaCl₂∙2H₂O, 0.37 g l⁻¹ MgSO₄∙7H₂O, 37.3 mg l⁻¹ Na₂-EDTA, 27.8 mg l⁻¹ FeSO₄∙7H₂O, 0.2 mg ml⁻¹ KH₂PO₄, 0.1 mg ml⁻¹ myo-inositol, 1 mg l⁻¹ thiamine-HCl, 0.2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid, and 30 g l⁻¹ sucrose [pH 5.8]; in this medium, the final Ca²⁺ concentration is 120 mg l⁻¹) containing 100 mg l⁻¹ kanamycin in a 300-ml conical flask with continuous light at 22°C with 100 rpm shaking and were subcultured weekly at 25-fold dilution.

Induction of xylem vessel cell differentiation and changes in medium Ca²⁺ concentration

To induce xylem vessel cell differentiation, dexamethasone (DEX) (final concentration 1 µM) was applied to T87/35S::VND7-VP16-GR cells 3 days after subculture. As the mock treatment, we treated cells with the equivalent amount of 99.5% ethanol (Wako). For experiments involving changes in medium Ca²⁺ concentration, the amount of CaCl₂∙2H₂O in the culture medium was changed by the replacement of medium, before the addition of DEX. Three-day-old T87/35S::VND7-VP16-GR cells were centrifuged at 800 g for 1 min, and the medium was discarded. The collected cells were placed in 10 ml medium with different Ca²⁺ concentrations. These steps were repeated three times. The final concentration of Ca²⁺ was set to 0, 50, 100, 120, 150, 200, 500, 1,000, or 5,000 mg l⁻¹. After the medium was changed, DEX was added. To evaluate xylem vessel cell differentiation, the number of cells with SCWs stained as described below was counted under the microscope, and the fraction of cells with SCWs was calculated and compared to the fraction of cells with SCWs in control samples without medium exchange (Figure 1).

Figure 1. Changes in medium Ca²⁺ concentration influence the efficiency of xylem vessel cell differentiation. Three-day-old T87/35S::VND7-VP16-GR cells were treated with dexamethasone (DEX) in medium containing the indicated concentration of Ca²⁺, and differentiation was assessed 24, 48, and 72 h after DEX treatment. Differentiation is shown relative to the fraction of cells with SCWs in control samples without medium exchange. Normal culture medium contains 120 mg l⁻¹ Ca²⁺. Data are presented as mean±SD (n=3).

Microscopic observation

DEX-treated cells were collected at 0, 24, 48, and 72 h of culture; fixed with 3.7% (v/v) formaldehyde solution; washed with phosphate-buffered saline (20 mM sodium phosphate buffer and 150 mM NaCl; pH 7.0) three times; and then stained with 0.001% (w/v) Calcofluor white or 0.01 mg ml⁻¹ WGA-Alexa594. Observation was performed using a fluorescence microscope (BX51, Olympus) or confocal laser scanning microscope (FLUOVIEW FV10i, Olympus). To evaluate cell morphology, the circularity, aspect ratio and solidity of cells were examined using ImageJ (Schneider et al. 2012).

Calcium-channel inhibitor treatment

Inhibitors were applied at the same time as DEX. The final concentrations of verapamil (FUJIFILM Wako Pure Chemical Industries, Ltd. Corporation) were 1, 3, 10, 100, and 1,000 µM, whereas those of bepridil (Cayman Chemical) and nifedipine (FUJIFILM Wako Pure Chemical Industries, Ltd. Corporation) were 1, 3, and 10 µM. To calculate the parallelness of SCWs, the fluorescence images were binarized and skeletonized using ImageJ (Schneider et al. 2012). Based on the skeletonized SCWs images, the parallelness was measured using the ImageJ plugin LpxLineFeature, as previously reported (Ueda et al. 2010).

Gene expression analysis

T87/35S::VND7-VP16-GR cells were collected after DEX treatment and immediately frozen in liquid nitrogen. Total RNA was extracted from crushed cell samples using the PureLink RNA Mini Kit (Thermo Fisher SCIENTIFIC) according to the manufacturer’s protocol. Total RNA samples were then treated by RQ1 DNase (Promega), followed by cDNA synthesis by Transcriptor Reverse transcriptase (Roche) using Oligo (dT)18 Primer (Roche) or Random Hexamer Primer (Roche). The synthesized cDNA was used as a template for PCR. PCR was performed with Quick Taq polymerase (TaKaRa) with conditions of 94°C for 2 min; 30 cycles of 94°C for 30 s, 60°C for 30 s, and 68°C for 3 min; and a final extension at 68°C for 7 min. The PCR products were then stained with 0.5 mg l⁻¹ ethidium bromide. The primers for the VND7-downstream genes were described in Yamaguchi et al. (2011) and in Hirai et al. (2020).

Results

Changes in the medium Ca²⁺ concentration affect the efficiency of xylem vessel differentiation

Transdifferentiation of T87/35S::VND7-VP16-GR cells into xylem vessel cells can be effectively induced by DEX activation of VND7, a master transcription factor for xylem vessel formation (Yamaguchi et al. 2010). To investigate effects of changes in medium Ca²⁺ concentration on xylem vessel cell differentiation, we treated 3-day-old T87/35S::VND7-VP16-GR cells with DEX in medium containing different Ca²⁺ concentrations within the range of 0–5,000 mg l⁻¹ and observed them 0, 24, 48, and 72 h later. We confirmed that the increased medium Ca²⁺ concentration (220, 360, and 1,450 mg l⁻¹) did not affect the cell morphology of T87/35S::VND7-VP16-GR cells strongly (Supplementary Figure S1), and that changing the medium just before DEX treatment did not influence differentiation ratio (Figure 1) or SCW patterning (Supplementary Figure S2).

Increases in medium Ca²⁺ concentrations decreased the differentiation ratio (Figure 1) but did not affect SCW patterning in differentiated cells (Supplementary Figure S2). Differentiation was severely reduced by exposure to medium containing 0, 500, 1,000, or 5,000 mg l⁻¹ Ca²⁺ (Figure 1). These results indicate that differentiation into xylem vessel cells was greatly inhibited by extremely low (0 mg l⁻¹) or extremely high (500, 1,000, and 5,000 mg l⁻¹) extracellular Ca²⁺ concentrations.

Changes in medium Ca²⁺ concentration do not inhibit the induction of VND7-downstream genes

We examined whether the expression of VND7-downstream genes was affected by changes in medium Ca²⁺ concentration. We performed semi-quantitative RT-PCR analysis 24 h after DEX treatment for VND7-downstream genes including the MYB transcription factor genes MYB46 and MYB83, secondary master switches for SCW formation (Ko et al. 2009; McCarthy et al. 2009; Zhong et al. 2007); MYB63 for lignin biosynthesis (Nakano et al. 2010; Zhou et al. 2009); SCW-type cellulose synthase genes CesA7 and CesA8 (Pear et al. 1996; Taylor et al. 1999; Turner and Somerville 1997); hemicellulose synthesis gene IRX8 (Brown et al. 2005); lignin monomer synthesis gene CCoAOMT7 (Raes et al. 2003); and PCD-related xylem-specific protease genes XCP1 and MC9 (Bollhöner et al. 2013; Funk et al. 2002). The expression of these genes was not detected after mock treatment (Supplementary Figure S3), although the increased Ca²⁺ concentration triggered calcium response at gene expression levels (Supplementary Figure S4). By contrast, after DEX treatment, the expression of these genes was induced in media with all Ca²⁺ concentrations except 5,000 mg l⁻¹ Ca²⁺ (Figure 2). At 5,000 mg l⁻¹ Ca²⁺, only MYB46, MYB83, and CCoAOMT7 were upregulated (Figure 2), possibly because there was almost no differentiation at this Ca²⁺ concentration (Figure 1; Supplementary Figure S2). The observation that VND7-downstream genes were induced even at Ca²⁺ concentrations of 0, 500, and 1,000 mg l⁻¹, where differentiation was greatly reduced (Figure 1), suggests that the inhibition of xylem vessel cell differentiation by changes in extracellular Ca²⁺ concentration occurs mainly through post-transcriptional processes.

Figure 2. Changes in medium Ca²⁺ concentration do not inhibit upregulation of VND7-downstream genes. Three-day-old T87/35S::VND7-VP16-GR cells were treated with dexamethasone (DEX) in medium containing the indicated concentration of Ca²⁺ and sampled 24 h later. Total RNA extracted from these cells was subjected to semi-quantitative RT-PCR analysis for VND7-downstream genes. As the control (C), cells without medium exchange were used. Normal culture medium contains 120 mg l⁻¹ Ca²⁺. UBQ10 was tested as an internal control gene. The analysis was repeated three times as biological replicates.

Calcium-channel inhibitors affect SCW patterning

Next, we evaluated whether calcium channels are involved in the inhibitory effects of changes in extracellular Ca²⁺ concentration on differentiation. T87/35S::VND7-VP16-GR cells were treated with the L-type calcium-channel inhibitor verapamil in media with 0, 120, or 5,000 mg l⁻¹ Ca²⁺ (Figure 3). Verapamil treatment restored the differentiation rate in 5,000 mg l⁻¹ Ca²⁺ medium in a dose-dependent manner (Figure 3). This observation suggests that the inhibition of xylem vessel cell differentiation under high Ca²⁺ concentration is due, at least partly, to the intracellular influx of extracellular Ca²⁺ through calcium channels. High-concentration verapamil (100 µM) significantly reduced the rate of xylem vessel cell differentiation in 0 mg l⁻¹ Ca²⁺ medium (Figure 3). Hence, the influx of extracellular Ca²⁺, some of which are probably contained in the cell wall, through calcium channels is required for xylem vessel cell differentiation, as previously reported (Kobayashi and Fukuda 1994; Roberts and Haigler 1990).

Figure 3. The calcium-channel inhibitor verapamil affects the efficiency of xylem vessel cell differentiation. Three-day-old T87/35S::VND7-VP16-GR cells were treated with dexamethasone (DEX) in medium containing 0, 120, or 5,000 mg l⁻¹ Ca²⁺, supplemented with 0, 1, 10, or 100 µM verapamil, and differentiation was assessed 24 h after DEX treatment. Normal culture medium contains 120 mg l⁻¹ Ca²⁺, and the medium exchange did not affect the differentiation efficiency. Data are presented as mean±SD (n=3). * p<0.05 (Student’s t-test, compared with the results of 0 µM verapamil).

Abnormalities in SCW patterning were observed in the verapamil-treated cells compared with mock-treated cells in medium with normal Ca²⁺ concentration (120 mg l⁻¹) (Figure 4A–F). Mock-treated cells deposited SCWs with well-aligned and parallel bundled structures (Figure 4A–C). By contrast, verapamil-treated cells deposited SCW bundles with branches, and net-like SCW structures were often observed as a result (Figure 4D–F). Quantitative imaging analysis indicated a significant decrease in SCW parallelness in the verapamil-treated cells (Figure 4M). We observed similar abnormal SCW patterns in cells treated with additional calcium-channel inhibitors, namely bepridil and nifedipine (Figure 4G–M), whereas these calcium-channel inhibitors did not affect the cell morphology itself (Supplementary Figure S5). These observations demonstrate that proper regulation of the influx of extracellular Ca²⁺ by calcium channels is important for xylem vessel cell differentiation as well as SCW patterning.

Figure 4. Calcium-channel inhibitors affect SCW patterning. (A–L) Three-day-old T87/35S::VND7-VP16-GR cells were treated with dexamethasone (DEX), along with mock treatment (A–C), verapamil (D–E), bepridil (G–I), or nifedipine (J–L) for 3 day. Scale bar=10 µm. (M) Imaging analysis for parallelness of SCW bundles in the cells shown in (A–L) (n=40 in each condition). * p<0.05, ** p<0.001 (Student’s t-test, compared with the results of mock-treated cells).

Discussion

In this work, we investigated the contribution of Ca²⁺ signaling to xylem vessel cell differentiation using T87/35S::VND7-VP16-GR cells cultured in medium with different concentrations of Ca²⁺. Our results indicate that a certain amount of extracellular Ca²⁺ is required for xylem vessel cell differentiation, but excessive amounts of Ca²⁺ strongly inhibit differentiation (Figures 1, 3). Furthermore, extracellular Ca²⁺-dependent regulation of differentiation is mediated by calcium channels (Figure 3). These results are consistent with previous observations in zinnia (Groover and Jones 1999; Iakimova and Woltering 2017; Kobayashi and Fukuda 1994; Roberts and Haigler 1990). Thus, the importance of Ca²⁺ signaling in xylem vessel cell differentiation is conserved between Arabidopsis and zinnia.

In the T87/35S::VND7-VP16-GR system, the disturbance of Ca²⁺ signaling affects xylem vessel cell differentiation post-transcriptionally, based on our results showing that the expression of VND7-downstream genes was induced even under conditions that inhibited differentiation (0, 500, 1,000, and 5,000 mg l⁻¹; Figure 2). In eukaryotic cells, Ca²⁺/CaM signaling is important in the regulation of protein translation (Brostrom and Brostrom 1990). A critical piece of molecular evidence of the direct interaction between Ca²⁺ and protein synthesis is the multifunctional protein eukaryotic elongation factor 1α (eEF1A), whose canonical function is to deliver the aminoacyl-tRNA to the A-site of the 80S ribosome (Sasikumar et al. 2012). The eEF1A protein directly interacts with CaM and Ca²⁺/CaM-dependent protein kinase (Wang and Poovaiah 1999). Degradation of eEF1A is also regulated by Ca²⁺ (Ransom-Hodgkins et al. 2000). Proteome analysis of tobacco BY-2/35S::VND7-VP16-GR cells showed that the protein level of eEF2B, a nucleotide exchange factor required to reactivate eEF1A (Sasikumar et al. 2012), is increased after 24 h of DEX treatment (Noguchi et al. 2018), suggesting upregulation of translational activity. It is possible that the translation of VND7-downstream proteins that are necessary for xylem vessel cell differentiation is tightly regulated by Ca²⁺/CaM signaling. Early work in zinnia demonstrated that CaM function is required at 44–66 h of induction, just before the start of SCW formation (Kobayashi and Fukuda 1994), and that the VND7 homologue Z567 is upregulated before this, after 24 h of induction (Demura et al. 2002). These observations are in good agreement with our hypothesis that protein synthesis of VND7-downstream factors is regulated by Ca²⁺/CaM.

We also observed that calcium-channel inhibitors disturbed SCW patterning during xylem vessel cell differentiation (Figure 4). The patterned deposition of the major SCW components cellulose, xylan, and lignin is regulated by cortical microtubules (Oda et al. 2015; Takenaka et al. 2018; Watanabe et al. 2015; Wightman and Turner 2010). For example, cellulose is produced by a cellulose synthase (CesA) complex (CSC) located on the plasma membrane (Wightman and Turner 2010). CSCs are transported to the plasma membrane via the trans-Golgi network and the endoplasmic reticulum close to the plasma membrane, through the guidance of microtubules and F-actin (Haigler and Brown 1986; Wightman and Turner 2010). Guided by cortical microtubules, CSCs generate cellulose microfibrils to the extracellular space (Gutierrez et al. 2009; Paredez et al. 2006). Notably, Ca²⁺ and CaM affect the structure and dynamics of both the actin and microtubule cytoskeleton (Hepler 2016; Kölling et al. 2019). Recent findings regarding Ca²⁺- and CaM-interacting microtubule-associated proteins (MAPs) have revealed further roles for Ca²⁺/CaM signaling in regulating the plasma membrane and cytoskeleton in plant cells (Kölling et al. 2019). In this paper, we first reported the effects of calcium-channel inhibitors on SCW patterning. Our observations of abnormal SCW patterns (Figure 4) point to Ca²⁺/CaM-dependent regulation of microtubules during xylem vessel cell differentiation. Notably, CaM proteins are upregulated during xylem vessel cell differentiation in zinnia (Kobayashi and Fukuda 1994; Roberts and Haigler 1990), and recently, the xylem-specific CaM protein PdIQD10 was reported to bind MAP and regulate SCW formation in Populus deltoides (Badmi et al. 2018). Further identification and functional analysis of xylem cell-specific CaM proteins should provide important molecular clues to understand and manipulate the Ca²⁺/CaM signaling pathway in xylem vessel cell differentiation.

Calcium is one of important molecules for eukaryotes to mediate intracellular and intercellular signaling. In the present work, we showed that the importance of extracellular Ca²⁺ condition for xylem vessel cell differentiation (Figure 1). Since the Ca²⁺ influx into the cells changes cytosolic Ca²⁺ immediately and greatly, the active regulation of Ca²⁺ influx would be an effective strategy for plant cells to sense the biotic and abiotic stresses outside the cell (Hepler 2005); indeed, the influx of extracellular Ca²⁺ through the activation of calcium channels is one of the first critical steps during plant disease response (Wang et al. 2019). Thus, plants might control xylem vessel cell differentiation in response to the extracellular Ca²⁺ condition, and/or the activity of calcium channels, to adapt the conductive functions of xylem vessels to the environments. Further elucidation of Ca²⁺/CaM-dependent regulatory mechanism of xylem vessel cell differentiation would make clear novel strategy of plant environmental adaptation.

Abbreviations

Ca²⁺

calcium ion

DEX

dexamethasone

NAC

NAM/ATAF/CUC

PCD

programmed cell death

SCW

secondary cell wall

VND

VASCULAR-RELATED NAC-DOMAIN