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. 2020 Feb 5;182(4):2199–2212. doi: 10.1104/pp.19.00784

Callose Synthesis Suppresses Cell Death Induced by Low-Calcium Conditions in Leaves1

Yusuke Shikanai a,2, Ryosuke Yoshida a,2,3, Tomoko Hirano a,2,4, Yusuke Enomoto a,5, Baohai Li a,6, Mayu Asada a,7, Mutsumi Yamagami b, Katsushi Yamaguchi c, Shuji Shigenobu c, Ryo Tabata d, Shinichiro Sawa e, Hiroki Okada f,8, Yoshikazu Ohya f, Takehiro Kamiya a,g, Toru Fujiwara a,9,10
PMCID: PMC7140939  PMID: 32024698

Callose is required for the suppression of cell death and necrosis induced by calcium deficiency.

Abstract

Despite the importance of preventing calcium (Ca) deficiency disorders in agriculture, knowledge of the molecular mechanisms underlying plant adaptations to low-Ca conditions is limited. In this study, we provide evidence for a crucial involvement of callose synthesis in the survival of Arabidopsis (Arabidopsis thaliana) under low-Ca conditions. A mutant sensitive to low-Ca conditions, low calcium sensitive3 (lcs3), exhibited high levels of cell death in emerging leaves and had defects in its expanding true leaves under low-Ca conditions. Further analyses showed that the causal mutation was located in a putative β-1,3-glucan (callose) synthase gene, GLUCAN SYNTHASE-LIKE10 (GSL10). Yeast complementation assay results showed that GSL10 encodes a functional callose synthase. Ectopic callose significantly accumulated in wild-type plants under low-Ca conditions, but at a low level in lcs3. The low-Ca sensitivity of lcs3 was phenocopied by the application of callose synthase inhibitors in wild-type plants, which resulted in leaf expansion failure, cell death, and reduced ectopic callose levels under low-Ca conditions. Transcriptome analyses showed that the expression of genes related to cell wall and defense responses was altered in both wild-type plants under low-Ca conditions and in lcs3 under normal-Ca conditions, suggesting that GSL10 is required for the alleviation of both cell wall damage and defense responses caused by low Ca levels. These results suggest that callose synthesis is essential for the prevention of cell death under low-Ca conditions and plays a key role in plants’ survival strategies under low-Ca conditions.

Calcium (Ca) is an important macronutrient in plants. Plants require Ca for many processes, including cell wall and plasma membrane stabilization, enzyme activation, and signal transduction (Marschner, 2011). These functions are important in developing or growing tissues. Typical Ca deficiency symptoms include blossom-end rot in growing tomato (Solanum lycopersicum) fruits and tipburn in expanding leaves of Chinese cabbage (Brassica rapa ssp. pekinensis; Kuo et al., 1981; White and Broadley, 2003). In general, plants exhibit deficiency symptoms if the concentration of an essential element in the soil or their tissues is insufficient. Under Ca deficiency (0.29 mm Ca), 53% of tomato fruit were found to develop blossom-end rot; in contrast, less than 10% of tomato fruit were affected when grown under 2.9 mm Ca (Schmitz-Eiberger et al., 2002). Similarly, a susceptible inbred line of Chinese cabbage developed tipburn under 0.57 mm Ca but showed healthy growth under 5.7 mm Ca (Su and Yu et al., 2016).

Ca makes up 3.64% of the earth’s crust and is a component of minerals such as calcite, dolomite, and aragonite (Taylor and Locascio, 2004). In soil, Ca concentrations are relatively high (10–60 mm according to Stein et al. [2017], with estimations of soil porosity and water contents of 50% and 30%, respectively). However, Ca deficiency symptoms often occur in agriculture (Taylor and Locascio, 2004) because Ca is mainly translocated by the transpiration stream, and conditions that affect transpiration can cause Ca deficiency as well as reduce Ca concentrations in soil. For example, in tomato, the transport of Ca to young leaves has been reported to be reduced under high humidity relative to low humidity (Armstrong and Kirkby, 1979). Thus, Ca fertilization of the soil alone is not sufficient to prevent Ca deficiency symptoms (Olle and Bender, 2009).

As Ca is translocated via the transpiration stream, it tends to accumulate more in older, expanded leaves and less in rapidly growing tissues such as new, small leaves, where the demand for Ca to provide new cell wall materials is high (Bangerth, 1979). The Ca deficiency symptoms occur in rapidly growing tissues such as new leaves and fruits (White and Broadley, 2003), which is thought to be caused by an imbalance between the supply of and demand for Ca among tissues.

Breeding of crops that are tolerant to low-Ca conditions is an alternative and promising strategy to prevent the development of Ca deficiency symptoms. For the efficient molecular breeding of low-Ca-tolerant crops, identifying the genes involved in the development of Ca deficiency symptoms and/or low-Ca tolerance is important. Several genes have been reported to be involved in the maintenance of growth under low-Ca conditions. For example, the overexpression and disruption of a vacuolar Ca2+/H+ antiporter, CAX1, was found to enhance Ca deficiency symptoms (tipburn) in tobacco (Nicotiana tabacum; Hirschi, 1999) and low-Ca tolerance in the shoots of Arabidopsis (Arabidopsis thaliana; Cheng et al., 2003), respectively. The disruption of endoplasmic reticulum Ca2+-ATPase also led to reduced shoot growth under low-Ca conditions in Arabidopsis (Wu et al., 2002). A double mutation of spermine synthase resulted in reduced shoot growth under low-Ca conditions in Arabidopsis (Yamaguchi et al., 2006). The overexpression of an endoplasmic reticulum Ca2+-binding protein, CRT1, was previously found to alleviate Ca deficiency symptoms (blossom-end rot in tomato and tipburn in tobacco) caused by the overexpression of CAX1 (Wu et al., 2012). The down-regulation of pectin methyl esterase by RNA interference resulted in the alleviation of blossom-end rot in tomato (de Freitas et al., 2012). However, the physiological strategies used by plants to overcome periods of low Ca availability, especially the mechanisms that prevent necrotic lesion formation in commercially important parts of crops, such as the fruits or leaves, are not yet known.

The mechanisms used by plants to overcome Ca deficiency have been elucidated by isolating and analyzing Arabidopsis mutants sensitive to low-Ca conditions (Shikanai et al., 2015; Li et al., 2017). In this study, we used such mutants and showed that β-1,3-glucan (callose) synthesis is required for the prevention of cell death under low-Ca conditions and that it constitutes an essential part of low-Ca tolerance mechanisms in plants.

RESULTS

True Leaves of low-calcium sensitive3 Exhibited Necrosis and Did Not Expand under Low-Ca Conditions

To understand the low-Ca tolerance mechanisms of plants, we screened ethyl methanesulfonate-mutagenized Arabidopsis plants (Columbia-0 [Col-0]) to isolate mutants hypersensitive to low-Ca conditions. One of these mutants, low-calcium sensitive3 (hereafter referred to as lcs3), exhibited strong growth defects, and almost all plants developed no true leaves under the low-Ca (0.2 mm) condition tested, whereas lcs3 could apparently grow normally, although with slightly reduced shoot growth (85%–95% relative to that of Col-0 plants), under the normal-Ca (2 mm) condition used (Fig. 1, A and B; Supplemental Fig. S1A). The lcs3 mutant showed sensitivity to not only low Ca but also excess magnesium (Mg), suggesting that the causal gene of lcs3 may contribute to tolerance to the condition of serpentine soil, which is regarded to have a high Mg/Ca ratio (Supplemental Fig. S2; Walker et al., 1955).

Figure 1.

Figure 1.

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Growth inhibition and high frequency of cell death exhibited by lcs3 under low-Ca conditions. A, Phenotypes of Col-0 and lcs3 plants. Plants were grown under normal-Ca (2 mm) and low-Ca (0.2 mm) conditions for 14 d. Bars = 1 cm. B, The number of true leaves of 14-d-old seedlings of Col-0 and lcs3 plants. Values represent means ± sd. Asterisks indicate significant differences between Col-0 and lcs3 plants under each Ca condition (Student’s t test, two-tailed P < 0.05; n = 12–14). C, Trypan Blue staining of the first and second true leaves of Col-0 and lcs3 plants. Plants were grown with 2 mm Ca for 5 d, transferred to 2 or 0.2 mm Ca, and incubated for 2 d. Bars = 200 µm. D, Percentage of lesion area per leaf area. Plants were grown in the same conditions as in C. The first and second true leaves from 10 independent plants (n = 20) were used for lesion area quantification. Whiskers represent the smallest or largest value whose distance from the first quantile or third quantile does not exceed 1.5-fold of the interquantile range, respectively. Different letters indicate significant differences (Steel-Dwass test, P < 0.05).

Ca deficiency symptoms such as blossom-end rot in tomato or tipburn in Chinese cabbage are known to result in the development of cell death lesions in newly developing tissues. To determine whether the low-Ca sensitivity of lcs3 was associated with cell death, we visualized the dead cells in its leaves (Fig. 1C). Col-0 and lcs3 plants were grown under the normal-Ca (2 mm) condition for 5 d, transferred to either normal- or low-Ca (0.2 mm) conditions for 2 d, and then subjected to Trypan Blue staining. Trypan Blue stains dead cells blue (Koch and Slusarenko, 1990). We found that the low-Ca condition increased the area of dead cells in lcs3 leaves, which was larger than that in the leaves of Col-0 plants (Fig. 1, C and D). Moreover, lcs3 directly sown and grown under the low-Ca condition showed cell death throughout the new leaves, including the basal portions that supply new cells to the growing leaves (Supplemental Fig. S1B; Gonzalez et al., 2012). These results indicated that the low-Ca treatment induced cell death in leaves and that lcs3 failed to limit the occurrence of cell death under the low-Ca condition in new leaves.

We also performed transfer experiments to investigate whether the change in Ca condition during growth affected the development of deficiency symptoms. Col-0 and lcs3 were grown under normal Ca for 7 d and then transferred to normal- or low-Ca conditions and incubated for 7 d (Supplemental Fig. S3, A and B). The lcs3 plants transferred to low Ca showed significantly reduced shoot fresh weight. Furthermore, a 3-d exposure of normally grown lcs3 plants to low Ca induced brown lesion formation in true leaves (Supplemental Fig. S3, C and D). These results suggest that lcs3 exhibits low Ca sensitivity after exposure to low Ca during growth. We found that the cells in the brown lesions in the true leaves were smaller than those in the surrounding area (Supplemental Fig. S3, E–H), suggesting that cell death occurred during cell expansion, consistent with the findings of previous studies suggesting that necrosis during tipburn development occurs in the tip region of leaves where cells are expanding (Saure, 1998).

The Ca concentration in the leaves was not significantly different between Col-0 and lcs3 plants under the normal- and mildly low-Ca (0.4 mm) conditions, according to the inductively coupled plasma-mass spectrometry analysis (Supplemental Fig. S1C; Supplemental Methods). The concentrations of other elements (boron, sodium, Mg, phosphorus, potassium, manganese [Mn], copper, and zinc) did not differ significantly under the mildly low-Ca condition between Col-0 and lcs3 (Supplemental Fig. S4, A and B), suggesting that element transport is not defective in the mutant. We also measured the rate of water loss to obtain insights into the effect of mutation on the transpiration stream (Supplemental Fig. S5). Col-0 and lcs3 were grown under 2 mm Ca for 7 d and then transferred to 2 and 0.2 mm Ca, followed by incubation for 7 d. Reduction of detached shoot weight was measured over time; no significant differences were found between Col-0 and lcs3 at both Ca conditions. Taken together, these results suggest that lcs3 does not have strong defects in Ca transport. We consider that the lcs3 mutation has stronger impacts on the utilization of Ca.

To clarify whether the low-Ca sensitivity of lcs3 was governed by processes in the shoot and/or roots, we performed grafting assays between Col-0 and lcs3 plants (Supplemental Methods). The frequency of necrotic lesions in new leaves of the Col-0/lcs3 (rootstock/scion) grafted plants was higher than that in the self-grafted Col-0 plants, whereas that in the lcs3/Col-0 (rootstock/scion) grafted plants was lower than that in self-grafted lcs3 plants (Supplemental Fig. S1D). These results indicated that the shoot genotype is responsible for the development of necrotic lesions in the leaves, further supporting the hypothesis that lcs3 has a defect in the efficiency of Ca usage within its local shoot tissues rather than in its efficiency of long-distance Ca transport.

The Causal Gene of lcs3 Is GLUCAN SYNTHASE-LIKE10

To identify the causal gene for the low-Ca sensitivity in lcs3, we performed map-based cloning by using simple sequence-length polymorphism markers in F2 crosses between lcs3 (Col-0 background) plants and Arabidopsis accession Landsberg erecta plants (Supplemental Fig. S6A). The causal mutation was found to be located between 0.2 and 3.1 Mb on chromosome 3. To identify the causal gene, we performed genome resequencing against the lcs3 sequence and found a mutation in the 47th intron-splicing site of AT3G07160, GLUCAN SYNTHASE-LIKE10 (GSL10; Fig. 2A). Reverse transcription PCR of the mutated site of GSL10 in lcs3 showed two aberrant transcripts in lcs3 (Supplemental Fig. S6B), both of which led to premature stop codons and the production of a truncated protein that lacks 11 of the amino acid residues of glucan synthase (β-1,3-glucan synthesis catalytic domain; Supplemental Fig. S7, A and B). Considering that the C-terminal-truncated GSL10 is transcribed in lcs3 (Supplemental Fig. S7, A and B), lcs3 was likely a mutant in which GSL10 was partially disrupted, which is consistent with the findings of a previous study showing that the complete disruption of GSL10 results in gametophyte lethality (Töller et al., 2008).

Figure 2.

Figure 2.

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The causal gene of lcs3 is GSL10, which encodes a functional callose synthase and contributes to callose accumulation under low-Ca conditions. A, Schematic gene model of GSL10/CalS9 (AT3G07160), the causal gene of the lcs3 mutation. The splice-site mutation resulting in the lcs3 and gsl10-6 mutations is shown. Black boxes, gray boxes, and bars indicate protein-encoding exons, untranslated regions, and introns, respectively. The annotated coding regions of the glucan synthase domain are shown as red boxes. WT, Wild type. B, Yeast complementation assay performed by introducing GSL10 under the control of a Gal-inducible promoter into the temperature-sensitive mutant fks1Δfks2 YOC1073 (Okada et al., 2010). FKS1 encodes a yeast β-1,3-glucan synthase. pRS316 and pYES2 are empty vector controls for FKS1 and GSL10, respectively. For this, 5 µL of yeast cultures was spotted with serial dilutions and then incubated for 5 d at 37°C. C, Quantification of callose accumulation in the cotyledons. The number of particles above the emission threshold per unit area is shown. Plants were grown for 5 d under each Ca condition. The number of analyzed plants is shown. Whiskers represent the smallest or largest value whose distance from the first quantile or third quantile does not exceed 1.5-fold of the interquantile range, respectively. Different letters indicate significant differences between Ca conditions (Steel-Dwass test, P < 0.05); n.s, no significant difference. D, Aniline Blue staining for callose in the cotyledons of plants of Col-0, gsl10-5, and complemented lines. Plants were grown under each Ca condition for 5 d and then fixed and subjected to Aniline Blue staining. DIC, Differential interference contrast. Bars = 200 µm.

To determine whether the causal gene of the lcs3 mutation is GSL10, we tested the sensitivity of a gsl10 allele (Arabidopsis Biological Resource Center stock no. CS92211) to low-Ca conditions. This line has a nonsynonymous mutation in GSL10 (G1682E). The low-Ca sensitivity of the CS92211 line was higher than that of Col-0 plants (Supplemental Fig. S6C). In addition, the introduction of the ProGSL10-GSL10cds-GFP construct (GSL10-GFP) into lcs3 partially complemented the growth inhibition and cell death lesion formation in new leaves that occurred under low-Ca conditions (Supplemental Fig. S6, D–F). Taken together, these results suggested that GSL10 is the causal gene of lcs3. Because four alleles of gsl10 have previously been reported (Huang et al., 2009), we renamed lcs3 as gsl10-5 and CS92211 as gsl10-6 and hereafter refer to them as such (Fig. 2A). The semidwarf phenotype of lcs3 plants at the flowering stage, which was also reported in the RNA interference line of GSL10 (Töller et al., 2008), was also complemented by the introduction of GSL10-GFP (Supplemental Fig. S6G).

To assess whether GSL10 expression patterns and levels were changed under low-Ca conditions, we performed promoter GUS assays and reverse transcription quantitative PCR (RT-qPCR) on plants grown under normal- and low-Ca conditions. No differences were found in the expression pattern and amount of this gene between the normal- and low-Ca conditions (Supplemental Fig. S8, A and B; Supplemental Methods), suggesting that GSL10 is expressed independently of Ca conditions. We also determined the subcellular localization of GSL10 by using the complemented line, GSL10-GFP, in gsl10-5 (Supplemental Fig. S8C; Supplemental Methods). The GFP signal was mainly observed around the cell surface, with a weak signal inside the cell, suggesting that GSL10 is mostly localized to the plasma membrane. In the SUBA3 subcellular localization database (http://suba.plantenergy.uwa.edu.au/), tandem mass spectrometry analysis detected GSL10 protein in the Golgi and plasma membrane in more than two independent experiments, supporting our findings.

GSL10 Encodes a Functional Callose Synthase

In the Arabidopsis genome, 12 GSL family members, which are all annotated as β-1,3-glucan (callose in plants) synthase, are present (Hong et al., 2001). Callose has long been known to be an essential cell wall polymer required for many biological processes, including cell division (Thiele et al., 2009), plasmodesmata regulation (Han et al., 2014), and plant immunity (Jacobs et al., 2003). A previous study showed that pollen from GSL10/gsl10-2 plants exhibited compromised callose deposition (Töller et al., 2008). However, whether GSL10 catalyzes β-1,3-glucan synthesis has not been tested in vivo. To validate the β-1,3-glucan synthase activity of GSL10, we performed a yeast (Saccharomyces cerevisiae) complementation assay by introducing the coding sequence of GSL10 under the control of a Gal-inducible promoter into a temperature-sensitive β-1,3-glucan synthase mutant (Okada et al., 2010). At a high temperature (37°C) with Gal, yeast carrying GSL10 showed improved growth compared with that of vector controls (Fig. 2B), suggesting that GSL10 encodes a functional β-1,3-glucan synthase. The results of these genetic analyses suggest that the low-Ca sensitivity of the gsl10-5 mutant is caused by defective callose synthesis.

Ectopic Callose Deposition Occurs under Low-Ca Conditions in Col-0 But Remains at a Low Level of Deposition in gsl10-5

Based on the results of the yeast complementation assay (Fig. 2B), we speculated that the gsl10-5 mutant exhibits reduced callose accumulation. Callose is known to be present under normal conditions as well as in response to environmental stresses such as pathogen infection. We defined callose deposited only under low-Ca conditions, which is not observed under normal-Ca conditions, as ectopic callose. To investigate whether ectopic callose accumulates in response to low-Ca treatment, we performed Aniline Blue staining of the cotyledons of Col-0, gsl10-5, and the complemented lines grown under conditions with 2 and 0.1 mm Ca for 5 d (Fig. 2, C and D). Under 2 mm Ca treatment, callose accumulation was low, and no significant difference was observed among any of the lines/genotypes tested (Fig. 2, C and D). Under the low-Ca (0.1 mm Ca) condition, ectopic callose accumulation was higher in Col-0 but lower in gsl10-5 (Fig. 2, C and D). The complemented lines of gsl10-5 showed the recovery of callose accumulation under the low-Ca condition (Fig. 2, C and D). These results indicated that ectopic callose accumulation occurs in response to low Ca. In addition, together with the results of yeast complementation analysis, these results indicated that GSL10 functions as a callose synthase in planta and is indispensable for ectopic callose accumulation under low-Ca conditions. Callose synthesis or deposition may therefore be important for low-Ca adaptation in Arabidopsis.

To further characterize callose deposition in response to low Ca, we compared the detailed location of callose depositions in wild-type plants between the 2 and 0.1 mm Ca conditions (Supplemental Fig. S9). Confocal images of samples stained with Aniline Blue revealed that callose was observed as dots with diameters in the range of 1 to 5 µm. Some of the dots were present between the cells (Supplemental Fig. S9A, blue arrowheads), and others were observed within the cells (Supplemental Fig. S9A, red arrowheads). These dot-like depositions were similar to the pattern of callose deposition in the cell wall matrix induced by defense responses after treatment with chitosan and/or Flg22 (Luna et al., 2011).

To further confirm the location of ectopic callose deposition, we performed whole-mount antibody staining by using an anti-callose antibody against the cotyledons of 4-d-old seedlings grown under 2 or 0.1 mm Ca conditions (Supplemental Methods). Dot-like structures within the cells were only visible under the low-Ca treatment, whereas signals in the region between the cells, indicative of plasmodesmata-associated callose depositions, were strongly detected irrespective of Ca conditions (Supplemental Fig. S9B). Our observation suggests that low Ca-induced ectopic callose accumulation occurs in either cell wall matrix or both cell wall matrix and plasmodesmata.

Inhibition of Callose Synthase Makes Col-0 Plants Sensitive to Low Ca

If callose synthesis is required for low-Ca adaptation, the inhibition of callose synthesis should make plants sensitive to low Ca levels. To test this hypothesis, we performed phenocopy assays by using 2-deoxy-d-glucose (DDG), which is used as a callose synthesis inhibitor (Jaffe and Leopold, 1984; Bayles et al., 1990). Col-0 and gsl10-5 plants were grown under different combinations of 2, 0.4, and 0.2 mm Ca and 0, 200, and 400 µm DDG (Fig. 3A). We found that the combination of a low Ca concentration and DDG (200 and 400 µm, respectively) reduced the number of leaves in Col-0 plants, suggesting that the inhibition of callose synthesis by DDG enhanced their low-Ca sensitivity (Fig. 3A). In addition, gsl10-5 mutants showed enhanced DDG sensitivity even after treatment with 2 mm Ca compared with that of Col-0 plants (Fig. 3A), suggesting that the gsl10-5 mutation has an additive effect with that of DDG application.

Figure 3.

Figure 3.

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Application of a callose synthase inhibitor enhanced the low-Ca sensitivity of Col-0 plants. A, The number of true leaves of Col-0 and gsl10-5 plants under the 2, 0.4, and 0.2 mm Ca conditions with or without the application of DDG. Plants were grown in the indicated conditions for 12 d. Whiskers represent the smallest or largest value whose distance from the first quantile or third quantile does not exceed 1.5-fold of the interquantile range, respectively. Different letters indicate significant differences among the Ca conditions (Steel-Dwass test, P < 0.05; n = 23–30). B, Trypan Blue staining of the first or second true leaves of Col-0 plants. Plants were grown at 2 mm Ca for 5 d, transferred to 0.2 mm Ca with 0 or 400 µm DDG, and incubated for 2 d. Bars = 500 µm. C, Percentage of lesion area per leaf area. Plants were grown in the same conditions as in B. For the quantification of lesion areas, 11 to 14 individual first or second true leaves from eight independent plants were used. Whiskers represent the smallest or largest value whose distance from the first quantile or third quantile does not exceed 1.5-fold of the interquantile range, respectively. Different letters indicate significant differences among treatments (Steel-Dwass test, P < 0.05).

Because cell death was enhanced in gsl10-5 plants under low-Ca conditions (Fig. 1C), we speculated that the application of DDG under low-Ca conditions would also enhance cell death in Col-0 plants. The results showed that cell death was enhanced by the combination of low Ca concentrations with DDG compared with the same Ca conditions without DDG (Fig. 3, B and C). Moreover, DDG treatment reduced the ectopic callose accumulation in true leaves under low-Ca conditions in Col-0 plants, similar to what was noted in gsl10-5 mutants (Supplemental Fig. S1, A and B), which is consistent with the findings of a previous study (Bayles et al., 1990). We also observed cell death in emerging leaves without further expansion of true leaves (Supplemental Fig. S11). Consistent with the results shown in Figure 1C, gsl10-5 mutants showed high levels of cell death in their new leaves, especially in the basal portions, after treatment with 0.2 mm Ca (Supplemental Fig. S11). Moreover, similar to gsl10-5 plants at 0.2 mm Ca, Col-0 plants with DDG treatments showed considerable cell death in the bases of the leaves, even in the presence of 2 mm Ca (Supplemental Fig. S11), suggesting that the inhibition of callose synthesis by DDG caused a similar pattern of cell death to that in gsl10-5 mutants. Taken together, these results confirm that callose synthesis is required for the prevention of cell death and subsequent necrosis under low-Ca conditions.

DDG is known to inhibit not only callose synthesis but also glycolysis. Therefore, this secondary effect of DDG could have possibly caused the poor leaf growth under low-Ca conditions in the Col-0 plants. To further confirm that the inhibition of callose (β-1,3-glucan) synthesis reduces shoot growth at low Ca concentrations, we used a different inhibitor of β-1,3-glucan synthase, caspofungin (Supplemental Fig. S12; Douglas, 2001). Caspofungin is known to noncompetitively inhibit β-1,3-glucan synthase, and, to our knowledge, no study has shown that it inhibits glycolysis. The combination of caspofungin treatment and low Ca concentrations reduced the shoot fresh weight of Col-0 plants compared with that when low-Ca conditions or caspofungin treatment were applied alone (Supplemental Fig. S12). This result further confirmed that the inhibition of callose synthesis is the main cause of the reduction of shoot growth noted under low-Ca conditions.

Defense Response Was Induced by Low-Ca Treatment as Well as by gsl10-5 Mutation

Our findings indicated that callose synthesis is essential for the prevention of necrosis in leaves under low-Ca conditions, which led us to investigate how the combination of the defect in callose synthesis and low Ca can cause cell death. To address this question as well as to investigate how transcriptomic profiles are changed under the low-Ca condition and in gsl10-5, we performed transcriptome analyses by using RNA sequencing (Fig. 4; Supplemental Spreadsheet S1).

Figure 4.

Figure 4.

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The transcriptome profile of gsl10-5 resembles those of low-Ca stress and defense responses. A, Venn diagram of genes with differential expression. The criterion for differential expression was q < 0.05 in exact tests. The low-Ca-responsive genes, gsl10-5 responsive genes, and the common genes of low-Ca-responsive genes and gsl10-5 responsive genes are indicated as blue, green, and pink, respectively. ∩, Intersection. B, Scatterplot of the 1,154 low-Ca-responsive genes and all genes, indicated as blue and grey, respectively. The x and y axes indicate the log2 fold change in Col-0 plants (low Ca/normal Ca) and under normal-Ca conditions (gsl10-5/Col-0), respectively. Spearman’s rank correlation coefficient (ρ) is shown. C, GO analysis of the 300 low-Ca-responsive and gsl10-5-responsive genes that are described in A. The results for the cellular component are shown. ER, Endoplasmic reticulum. D, Heat map of expression levels (log2 fold change versus Col-0, normal Ca) of SA-, JA-, and cell death-related genes in the reference genome set (for details, see “Materials and Methods”). Up- and down-regulated genes are expressed as red and blue, respectively. E, RT-qPCR of the SA and JA pathway marker genes. Relative mRNA accumulation levels (normalized to those of EF1a) are plotted on single logarithmic charts. Plants were grown with 2 mm Ca for 1 week, transferred to 2 or 0.2 mm Ca conditions, and incubated for another 1 week, and then total RNA was prepared from their shoots (n = 4 biological replicates). Asterisks indicate significant differences from expression in Col-0 plants under each Ca condition (Dunnett’s test, P < 0.05).

Col-0 and gsl10-5 were grown under normal Ca (2 mm) for 7 d and then transferred to normal- or low-Ca (0.2 mm) conditions for 7 d. Total RNA samples from their shoots were then used for RNA sequencing. We identified 1,154, 6,212, 553, and 3,802 differentially expressed genes (DEGs; q < 0.05) by querying a total of 33,602 genes and comparing the gene expression in four combinations: normal/low Ca in Col-0, normal/low Ca in gsl10-5, Col-0/gsl10-5 under normal Ca, and Col-0/gsl10-5 under low Ca, respectively (Fig. 4A). Numerous DEGs were found in normal/low Ca in gsl10-5, which may be related to the poor growth under low Ca (Supplemental Fig. S3, A and B).

To determine the relationship between the effect of low Ca and gsl10-5 mutation on the transcriptome, we compared DEGs in normal/low Ca in Col-0 and DEGs in Col-0/gsl10-5 under normal Ca. We found that 300 genes were commonly altered, indicating that more than half of the gsl10-5-responsive genes (300 of 553 genes: 54.2%) were affected by low Ca in Col-0 (Fig. 4A). Regarding Ca-responsive genes, substantial portions (about 25%: 300 of 1,154) were altered in gsl10-5. These results suggest that transcriptomic changes observed in gsl10-5 are substantially similar to those observed in Col-0 under the low-Ca condition. For further analysis, we generated a scatterplot between Col-0 plants under low-/normal-Ca conditions and in gsl10-5/Col-0 plants under the normal-Ca condition against all of the 33,602 genes and the 1,154 low-Ca-responsive genes (Fig. 4B). The plot showed a positive correlation (Spearman rank correlation coefficient of 0.41 for all genes and 0.815 for the low-Ca-responsive genes). These results indicated that low-Ca treatment and gsl10-5 mutation have similar impacts on the transcriptome, and this similarity is more evident in the low-Ca-responsive genes (Fig. 4B). Considering that callose synthesis is a Ca-dependent process (Him et al., 2001), the resemblance of the transcriptome pattern might be a reflection of the common reduction in the callose synthesis activity by either the low-Ca treatment or an effect of gsl10-5 mutation.

To further identify the genes that were commonly and differentially expressed between the low-Ca- and gsl10-5-responsive genes, we performed a Gene Ontology (GO) analysis against the 300 DEGs that were shared between the low-Ca- and gsl10-5-responsive gene groups (Fig. 4A). The most substantially enriched GO term among these genes in relation to cellular components was cell wall (Fig. 4C; Supplemental Fig. S13; Supplemental File), suggesting that the expression of cell wall-related genes was commonly and predominantly affected by both low Ca concentrations and the gsl10-5 mutation. Furthermore, the direction (up- or down-regulation; versus Col-0 under normal Ca) of the changes in the cell wall genes (43 genes in the 300 DEGs) was the same in the 40 genes of gsl10-5 under normal and 42 genes of Col-0 under low-Ca conditions (Supplemental Fig. S13). In addition, in gsl10-5 under low Ca, the extent of changes in 34 out of the 42 genes was enhanced. These results suggest that low Ca and gsl10-5 commonly affect cell wall homeostasis, at least in part.

Among the genes classified as related to cell wall in the GO analysis, several SA and JA signaling marker genes were included (Supplemental Fig. S14). The SA and JA signaling pathways are known to be activated as defenses against pathogen infection or wounding (Wildermuth et al., 2001; Bari and Jones, 2009). In addition, a correlation exists between the expression of SA signaling marker genes and cell death propagation under low Ca (Su and Yu et al., 2016). Taking these findings into consideration, our results suggest that defense responses are activated by low-Ca conditions as well as by the gsl10-5 mutation. In support of this interpretation, GO terms for response to abiotic or biotic stimulus and response to stress in biological processes were overrepresented among the 300 identified common genes (Supplemental Fig. S14). These results further indicate that defense responses are elicited by Ca deficiency and gsl10-5 mutation.

To investigate the effects of low Ca and gsl10-5 on the expression of SA, JA, and cell death-related genes in the transcriptome, we generated a heat map of the expression of genes annotated as SA, JA, and cell death in GO or functional descriptions (for the definition of these genes, see “Materials and Methods”). The direction of the change (up- or down-regulation) of the expression of these genes was common for the majority of the genes: for SA, 195 and 231 genes out of 292 genes examined were in the common direction in gsl10-5 under normal and low Ca, respectively; for JA, 219 and 245 genes out of 306, and for cell death-related genes, 54 and 58 genes out of 68 genes were in the common direction in gsl10-5 under normal and under low Ca, respectively. Furthermore, under low Ca, in gsl10-5, the extent of these expression changes was further enhanced in 195 out of 231, 205 out of 245, and 53 out of 58 SA-, JA-, and cell death-related genes, respectively, compared with that in Col-0 (Fig. 4E). These findings suggest that the effects of low Ca and gsl10-5 on SA and JA signaling and cell death are similar to each other and additive.

To further confirm the results of RNA sequencing, we performed RT-qPCR analysis on the expression of PR1 and PR2 (SA signaling marker genes) and PDF1.2A (a JA signaling marker gene; Mur et al., 2006) in gsl10-5 plants and complemented lines (Fig. 4E; Supplemental Spreadsheet S2; Supplemental Methods). The results showed that low-Ca conditions and gsl10-5 mutation induced the expression of some of these genes (PR1 and PR2), and the combination of low-Ca treatment and gsl10-5 mutation induced the expression of all the genes (PR1, PR2, and PDF1.2A), at considerably higher levels. Notably, PR2 encodes β-1,3-glucanase, a callose-degrading enzyme (BG2). We found that the expression of three BG genes, including PR2/BG2, was up-regulated among the low-Ca responsive genes (Supplemental Table S1); these three BG genes have been reported to be induced by fungal infection (Doxey et al., 2007). These results support the idea that the defense response, including SA and JA signaling, is induced by the low-Ca condition and gsl10-5 mutation, and their effects are synergistic.

We also determined the expression levels of callose- and pectin-related genes in our RNA sequencing data. No clear trend was noted in the alteration of the expression of GSL genes in response to low Ca (Supplemental Table S2). About pectin-related genes (PME, PMEI, GAUT, galacturonase, and galacturonase inhibitor), up-regulation of five PME genes and down-regulation of two pectin lyase genes were found among the 1,154 genes altered under low Ca in Col-0 (Supplemental Table S3).

DISCUSSION

In this study, we showed that GSL10 is required for the adaptation of Arabidopsis to low-Ca conditions (Fig. 2A; Supplemental Fig. S6, A–G) and that it encodes a functional callose synthase (Fig. 2B). GSL10 contributes to the suppression of cell death under low-Ca conditions (Fig. 1, C and D; Supplemental Fig. S6F). We also found that ectopic callose accumulation in response to low Ca levels is dependent on GSL10 (Fig. 2, C and D). Phenocopy assays performed using inhibitors showed that callose synthesis is essential for the expansion of leaves under low-Ca conditions (Fig. 3A; Supplemental Fig. S9). We showed that GSL10 is required for the alleviation of SA and JA defensive pathways elicited by low Ca levels (Fig. 4), suggesting that callose synthesis alleviates cell wall damage and defense responses induced by low Ca levels. Thus, we conclude that callose synthesis is a key strategy for plant survival under low-Ca conditions.

A conceptual diagram describing the role of callose synthesis in the prevention of cell death under low-Ca stress is shown in Figure 5. We hypothesize that low-Ca conditions induce cell wall damage, which is known to induce defense responses, including cell death, SA/JA pathway induction, and callose accumulation. In this diagram, callose accumulation alleviates cell wall damage, leading to SA/JA pathway suppression and cell death prevention. In gsl10-5 mutants, such callose accumulation would not be sufficient to alleviate cell wall damage under low-Ca conditions. The proposed cell wall damage induced by low Ca levels would strongly induce defense responses, including the SA/JA pathways and cell death, resulting in subsequent necrosis and failure of the expansion of leaves.

Figure 5.

Figure 5.

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Conceptual diagram of the low-Ca tolerance mechanism mediated by callose synthesis. Low Ca damages the cell wall, triggering defense responses such as cell death, SA/JA pathway activation, and callose accumulation. In Col-0, the accumulation of callose alleviates cell wall damage, enabling the suppression of cell death to a certain extent. In gsl10-5, because of the decrease of callose, the cell wall damage cannot be alleviated, resulting in necrosis in new leaves.

Possible Cell Wall Damage under Low-Ca Conditions

In this study, we revealed that both the expression of defense response (SA and JA) genes and the accumulation of ectopic callose were induced in response to low Ca concentrations (Figs. 2C and 4C; Supplemental Fig. S5). SA and JA pathway activation and callose accumulation have been regarded as components of plant defense responses to cell wall damage (Bari and Jones, 2009; Hamann et al., 2009). Thus, we proposed that low-Ca conditions induce cell wall damage. One of the most important functions of Ca in plant cell walls is to cross-link pectin (Powell et al., 1982). Low levels of Ca may reduce the formation of pectin-Ca cross-links and lead to cell wall damage. Indeed, fragments of pectin have been shown to function as damage-associated molecular patterns that induce defense responses, including callose accumulation (Denoux et al., 2008; Benedetti et al., 2015). Furthermore, Ca starvation has been reported to increase polygalacturonase (pectin-degrading) activity (Konno et al., 1984). The transcriptomic changes in pectin lyase genes in our RNA sequencing analysis (Supplemental Table S3) may reflect the possible physical changes in pectin under low-Ca conditions for the maintenance of pectin homeostasis. In addition, four out of five PME genes that were induced under low Ca have been reported to be involved in defense responses (Bethke et al., 2014), supporting our idea that low Ca causes cell wall damage and induces defense responses. Taken together, these results and those of previous studies suggest that low Ca levels induce cell wall damage.

Low-Ca-Induced Callose Inhibits Cell Death, Possibly through the Alleviation of Cell Wall Damage

We showed that, under low-Ca conditions, wild-type plants treated with a callose synthase inhibitor exhibited cell death in their new leaves, as was noted in gsl10-5 mutants without treatment with a callose synthase inhibitor (Fig. 3, B and C; Supplemental Fig. S10). These results indicate that callose synthesis can inhibit cell death under Ca-deficient conditions. Two previous studies performed using cell suspensions of Glycine max suggested that callose accumulation decreased the incidence of cell death (Kohle et al., 1984; Kauss and Jeblick, 1986). The first study showed that the cells treated with a callose synthesis inhibitor showed higher electrolyte leakage, an indicator of low plasma membrane integrity or cell death (Kauss and Jeblick, 1986). The second study suggested that callose accumulation decreased cell death rates after treatment with a cell wall-degrading enzyme (Kohle et al., 1984). Thus, the results of these previous studies suggest that callose accumulation alleviates cell wall damage and thus inhibits defense responses and cell death.

Callose synthesis has been shown to be dependent on Ca concentrations (Köhle et al., 1985; Him et al., 2001). Köhle et al. (1985) showed that about 95% of callose synthesis activity of suspension cells of G. max treated with chitosan was dependent on the Ca in the medium, while Him et al. (2001) showed that about 90% of callose synthase activity of Arabidopsis microsomes was dependent on Ca. Although a recent study showed the presence of Ca-independent callose synthesis activity in poplar (Populus spp.; Leijon et al., 2018), most of the callose synthesis activity in Arabidopsis was dependent on Ca. Cytosolic Ca has been shown to remarkably increase following influx of Ca from extracellular spaces (Köhle et al., 1985). In our study, callose accumulation was observed in response to low Ca levels in the medium, which is apparently inconsistent with the findings of these previous studies. This inconsistency can be explained by the activation of callose synthesis through Ca entering into the cytosol under low-Ca conditions. The Ca concentration in the extracellular space (the apoplast) is about 104-fold higher than that in the cytosol (Hepler, 2005). In general, the Ca concentration in the apoplast is maintained at a level of 1 to 10 mm (Hepler, 2005). In addition, a previous study showed that the minimum Ca concentration needed to induce callose accumulation was about 15 µm (Köhle et al., 1985). Therefore, Ca influx could still occur because of the extreme differences in Ca concentrations across the plasma membrane, even if the Ca conditions tested in our study (0.1–0.2 mm) would have decreased the Ca concentration in the apoplast. Our data showed that a 5-fold reduction in the Ca concentration in the culture medium resulted in less than 5-fold reduction in the Ca concentration of plant shoots (Supplemental Fig. S1C), supporting this idea. In addition, oligosaccharides from pectin (Denoux et al., 2008; Benedetti et al., 2015) might be involved in this activation.

Considering that callose accumulation is only observed under low-Ca conditions, but not under normal-Ca conditions, some factor(s) might promote callose synthesis when Ca concentrations are low. A possible factor promoting callose synthesis is cell wall damage. Indeed, as described above, cell wall damage is known to induce callose accumulation (Hamann et al., 2009). GSL10 mRNA accumulation was not remarkably induced by low Ca levels in this study (Supplemental Fig. S8B), which is also consistent with the involvement of cell wall damage in inducing Ca influx to activate GSL10 in a posttranslational manner.

Other Possible Roles of GSL10 and Callose in Low-Ca Tolerance

Although our results indicated that callose synthesis contributes to the suppression of cell death, GSL10 cannot be considered nonessential for the suppression of cell death in other ways than just through callose synthesis. In the gsl5 mutant, the expression of SA pathway genes was induced even under normal conditions, when callose accumulation was not detected in either wild-type or gsl5 plants (Nishimura et al., 2003; Ellinger et al., 2013). We also observed that the expression of defense response genes, including those involved in the SA pathway (Fig. 4, E and F), was enhanced in gsl10-5 plants in the normal-Ca condition, wherein little callose accumulation was observed in both wild-type and gsl10-5 plants (Fig. 2, C and D). Hence, the inhibition of growth in gsl10-5 plants under low-Ca conditions might be enhanced by the defense response(s) elicited by both the mutation in GSL10 and low Ca levels.

Other cell wall polymers may affect callose-mediated low-Ca tolerance. In this study, we demonstrated the accumulation of callose in response to low Ca. Considering that the cell wall is a complex structure composed of many different kinds of molecules (e.g. cellulose, pectin, and xyloglucan), other molecules might also be involved in the mechanism of callose-mediated low-Ca tolerance. In our transcriptome analysis, three xyloglucan endotransglycosylases/hydrolases were induced commonly by low Ca and gsl10-5 (Supplemental Fig. S13). This result suggests that the modification of xyloglucan is also affected by callose-mediated low-Ca tolerance. In addition, a recent study revealed the physical interaction between callose and cellulose (Abou-Saleh et al., 2018), supporting the idea that callose accumulation affects the entire system of the cell wall. Further studies are required to address this issue.

CONCLUSION

In conclusion, we showed that callose synthesis is a key low-Ca tolerance strategy in plants. Our findings reveal a mechanism of plant response to low Ca levels that prevents cell death and subsequent necrosis through metabolic changes and, in particular, the induction of callose synthesis. Ca deficiency symptoms include necrotic lesions in commercially important parts of crops, such as the new leaves or fruits. We expect that knowledge of this mechanism of low-Ca tolerance via callose synthesis can contribute to improved understanding of the development of necrosis in plants and to the efficient breeding of low-Ca-tolerant crops.

MATERIALS AND METHODS

Plant and Yeast Growth Conditions

For phenotyping analysis, surface-sterilized Arabidopsis (Arabidopsis thaliana) seeds were sown on medium plates containing nutrients (Yamagami medium; Shikanai et al., 2015) with 1% (w/v) Suc (Sigma-Aldrich, code 84097-250G) and 1.5% (w/v) agarose (Nacalai Tesque, code 01056-15). After seeds were incubated at 4°C for 2 to 7 d, plates were set vertically in a growth chamber for vernalization (photoperiod, 16 h of light and 8 h of dark) at 22°C. For phenocopy assays, plants were grown on the Yamagami medium in petri dishes with 1% (w/v) Suc and 0.7% (w/v) agarose containing Ca and an inhibitor, either DDG (Alfa Aesar, code L07338) or caspofungin acetate (Cayman, item no. 15923), for 12 d. The number of plants used for each assay is mentioned in the figures and figure legends.

For yeast (Saccharomyces cerevisiae) complementation assays, yeast strains (YOC1073 [MATa ade2 his3 leu2 lys2 trp1 ura3 fks1::HIS3 fks2::LYS2 ade3::fks1-1082:TRP1]; Okada et al., 2010) carrying plasmids were inoculated into 2 mL of liquid yeast extract peptone dextrose medium and incubated for 3 h at 27°C. Yeast cells were collected by centrifugation and suspended with 3 mL of liquid-selective medium (AHCW/Glc; Ueoka-Nakanishi et al., 2000) and then incubated for 3 d at 27°C. Next, 5 µL of fully grown yeast was inoculated into 3 mL of liquid AHCW/Glc and incubated overnight. Yeast cells from log-phase cultures (with an OD600 < 1) were collected by centrifugation, resuspended in distilled water, and adjusted to achieve an OD600 = 1. Subsequently, 5 µL of yeast was then spotted after serial 10-fold dilutions (designed to achieve OD600 = 1, 0.1, 0.01, and 0.001) on solid AHCW/Glc (2% [w/v] as the control condition) and AHCW/Gal and raffinose (2% and 1% [w/v], respectively, as the Gal-inducible condition) for 5 d at 37°C.

Trypan Blue Staining and Quantification

Trypan Blue staining was performed as described previously by Koch and Slusarenko (1990). The whole shoot was incubated in lactophenol Trypan Blue solution (lactic acid:phenol:Trypan Blue:glycerol:ethanol:distilled water = 5 mL:5 g:5 mg:5 mL:20 mL:5 mL) at 90°C for 5 min, and then the samples were transferred to chloral hydrate (chloral hydrate:distilled water = 5:2 [w/w]), incubated at 27°C overnight, and photographed.

For quantitative analysis, Fiji software (Schindelin et al., 2012) was used. The leaf area was measured using the Polygon Selections function in Fiji, and then the resulting polygons were cropped to generate image files showing one leaf each for further analysis. Cell death spots were identified by setting a color threshold for each observation to extract the area of stained cells and total stained area for use in further calculations.

Aniline Blue Staining, Observation, and Quantification

The seedlings of Arabidopsis were incubated in a fixative solution (50% [v/v] methanol and 10% [v/v] acetic acid; Truernit et al., 2008) at 4°C overnight or longer. Samples were then transferred to 80% (v/v) ethanol, incubated at 80°C for 5 min, transferred to chloral hydrate (chloral hydrate:distilled water = 5:2 [w/w]), and incubated at 27°C overnight. Next, the samples were incubated with 1 m Gly (pH 9.5) at 4°C for at least 3 h, transferred to 0.1 mg mL−1 Aniline Blue (Wako, code 016-21302) in 1 m Gly (pH 9.5), incubated for 2 h, mounted on glass slides with a mixture of glycerol and 1 m Gly (pH 9.5; 1:1 [v/v]), and then observed with a confocal microscope (Olympus, Fluoview FV1000). The excitation wavelength used was 405 nm, and the emission was measured between 480 and 530 nm. For quantification, images were obtained using optical depth set to that of the vascular bundles and then processed using Fiji (https://fiji.sc/; Schindelin et al., 2012) to count the number of particles in the image above the threshold emission level by using the function Analyze Particles. The threshold was set to be above the autofluorescence of dead cells, which was judged based on the samples not stained with Aniline Blue in each observation.

RNA Sequencing and Statistical Analyses

Seeds were sown on medium with normal Ca and grown for 1 week, and then transferred to medium with either normal or low Ca and incubated for another 1 week. Total RNA samples were then extracted from the shoots by using an appropriate kit (Nucleospin RNA plant; Takara) and used for RNA sequencing at BGI. SAM files were generated from fastq files by using TopHat2 (Trapnell et al., 2009) in the DDBJ pipeline (Nagasaki et al., 2013), and count files were generated using HTSeq (Anders et al., 2015) by using Python. Count files of eight libraries, which consisted of those from four test conditions (Col-0 under the normal-Ca condition, Col-0 under the low-Ca condition, gsl10-5 under the normal-Ca condition, and gsl10-5 under the low-Ca condition) with two biological replicates each, were normalized using the TMM methods (Robinson and Oshlack, 2010) by using the count per million method of edgeR (Robinson et al., 2010) in the R program (R Core Team, 2018). For GO analysis, Classification SuperViewer Tool w/Bootstrap was used (http://bar.utoronto.ca/ntools/cgi-bin/ntools_classification_superviewer.cgi; Provart and Zhu, 2003).

Definitions of SA-, JA-, and Cell Death-Related Genes

We selected genes from two data sets in TAIR (https://www.arabidopsis.org/download/index.jsp): ATH_GO_GOSLIM.txt and TAIR10_functional_descriptions.txt. SA-related genes and JA-related genes were the genes annotated as salicylic or jasmonic in the fifth column of ATH_GO_GOSLIM.txt or the genes annotated as salicylic or jasmonic in the Curator_summary column of TAIR10_functional_descriptions.txt. Cell death-related genes were the genes annotated as GO:0008219 (cell death) in the fifth column in ATH_GO_GOSLIM.txt or the genes annotated as cell death in the Curator_summary of TAIR10_functional_descriptions.txt.

Accession Numbers

RNA sequencing data described in this article have been deposited in the DNA Data Bank of Japan (accession no. DRA008360).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank Yuko Kawara and Emiko Yokota for excellent technical assistance and the Arabidopsis Biological Resource Center for providing the CS92211 line.

Footnotes

1

This work was supported by Japan Society for the Promotion of Science (grant no. 17J06965 to Y.S., grant no. 17H03782 to T.K., and grant nos. 15H01224, 18H05490, 19H05637, and 25221202 to T.F.)

References

  1. Abou-Saleh RH, Hernandez-Gomez MC, Amsbury S, Paniagua C, Bourdon M, Miyashima S, Helariutta Y, Fuller M, Budtova T, Connell SD, et al. (2018) Interactions between callose and cellulose revealed through the analysis of biopolymer mixtures. Nat Commun 9: 4538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anders S, Pyl PT, Huber W (2015) HTSeq: A Python framework to work with high-throughput sequencing data. Bioinformatics 31: 166–169 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Armstrong MJ, Kirkby EA (1979) The influence of humidity on the mineral composition of tomato plants with special reference to calcium distribution. Plant Soil 52: 427–435 [Google Scholar]
  4. Bangerth F. (1979) Calcium-related physiological disorders of plants. Annu Rev Phytopathol 17: 97–122 [Google Scholar]
  5. Bari R, Jones JD (2009) Role of plant hormones in plant defence responses. Plant Mol Biol 69: 473–488 [DOI] [PubMed] [Google Scholar]
  6. Bayles CJ, Ghemawat MS, Aist JR (1990) Inhibition by 2-deoxy-d-glucose of callose formation, papilla deposition, and resistance to powdery mildew in an mlo barley mutant. Physiol Mol Plant Pathol 36: 63–72 [Google Scholar]
  7. Benedetti M, Pontiggia D, Raggi S, Cheng Z, Scaloni F, Ferrari S, Ausubel FM, Cervone F, De Lorenzo G (2015) Plant immunity triggered by engineered in vivo release of oligogalacturonides, damage-associated molecular patterns. Proc Natl Acad Sci USA 112: 5533–5538 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bethke G, Grundman RE, Sreekanta S, Truman W, Katagiri F, Glazebrook J (2014) Arabidopsis PECTIN METHYLESTERASEs contribute to immunity against Pseudomonas syringae. Plant Physiol 164: 1093–1107 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cheng NH, Pittman JK, Barkla BJ, Shigaki T, Hirschi KD (2003) The Arabidopsis cax1 mutant exhibits impaired ion homeostasis, development, and hormonal responses and reveals interplay among vacuolar transporters. Plant Cell 15: 347–364 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. de Freitas ST, Handa AK, Wu Q, Park S, Mitcham EJ (2012) Role of pectin methylesterases in cellular calcium distribution and blossom-end rot development in tomato fruit. Plant J 71: 824–835 [DOI] [PubMed] [Google Scholar]
  11. Denoux C, Galletti R, Mammarella N, Gopalan S, Werck D, De Lorenzo G, Ferrari S, Ausubel FM, Dewdney J (2008) Activation of defense response pathways by OGs and Flg22 elicitors in Arabidopsis seedlings. Mol Plant 1: 423–445 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Douglas CM. (2001) Fungal β(1,3)-D-glucan synthesis. Med Mycol 39(Suppl 1): 55–66 [DOI] [PubMed] [Google Scholar]
  13. Doxey AC, Yaish MW, Moffatt BA, Griffith M, McConkey BJ (2007) Functional divergence in the Arabidopsis β-1,3-glucanase gene family inferred by phylogenetic reconstruction of expression states. Mol Biol Evol 24: 1045–1055 [DOI] [PubMed] [Google Scholar]
  14. Ellinger D, Naumann M, Falter C, Zwikowics C, Jamrow T, Manisseri C, Somerville SC, Voigt CA (2013) Elevated early callose deposition results in complete penetration resistance to powdery mildew in Arabidopsis. Plant Physiol 161: 1433–1444 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gonzalez N, Vanhaeren H, Inzé D (2012) Leaf size control: Complex coordination of cell division and expansion. Trends Plant Sci 17: 332–340 [DOI] [PubMed] [Google Scholar]
  16. Hamann T, Bennett M, Mansfield J, Somerville C (2009) Identification of cell-wall stress as a hexose-dependent and osmosensitive regulator of plant responses. Plant J 57: 1015–1026 [DOI] [PubMed] [Google Scholar]
  17. Han X, Hyun TK, Zhang M, Kumar R, Koh EJ, Kang BH, Lucas WJ, Kim JY (2014) Auxin-callose-mediated plasmodesmal gating is essential for tropic auxin gradient formation and signaling. Dev Cell 28: 132–146 [DOI] [PubMed] [Google Scholar]
  18. Hepler PK. (2005) Calcium: A central regulator of plant growth and development. Plant Cell 17: 2142–2155 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Him JL, Pelosi L, Chanzy H, Putaux JL, Bulone V (2001) Biosynthesis of (1→3)-β-d-glucan (callose) by detergent extracts of a microsomal fraction from Arabidopsis thaliana. Eur J Biochem 268: 4628–4638 [DOI] [PubMed] [Google Scholar]
  20. Hirschi KD. (1999) Expression of Arabidopsis CAX1 in tobacco: Altered calcium homeostasis and increased stress sensitivity. Plant Cell 11: 2113–2122 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hong Z, Delauney AJ, Verma DPS (2001) A cell plate-specific callose synthase and its interaction with phragmoplastin. Plant Cell 13: 755–768 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huang L, Chen XY, Rim Y, Han X, Cho WK, Kim SW, Kim JY (2009) Arabidopsis glucan synthase-like 10 functions in male gametogenesis. J Plant Physiol 166: 344–352 [DOI] [PubMed] [Google Scholar]
  23. Jacobs AK, Lipka V, Burton RA, Panstruga R, Strizhov N, Schulze-Lefert P, Fincher GB (2003) An Arabidopsis callose synthase, GSL5, is required for wound and papillary callose formation. Plant Cell 15: 2503–2513 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jaffe MJ, Leopold AC (1984) Callose deposition during gravitropism of Zea mays and Pisum sativum and its inhibition by 2-deoxy-D-glucose. Planta 161: 20–26 [DOI] [PubMed] [Google Scholar]
  25. Kauss H, Jeblick W (1986) Influence of free fatty acids, lysophosphatidylcholine, platelet-activating factor, acylcarnitine, and echinocandin B on 1,3-β-d-glucan synthase and callose synthesis. Plant Physiol 80: 7–13 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Koch E, Slusarenko A (1990) Arabidopsis is susceptible to infection by a downy mildew fungus. Plant Cell 2: 437–445 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Köhle H, Jeblick W, Poten F, Blaschek W, Kauss H (1985) Chitosan-elicited callose synthesis in soybean cells as a Ca-dependent process. Plant Physiol 77: 544–551 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kohle H, Young DH, Kauss H (1984) Physiological changes in suspension-cultured soybean cells elicited by treatment with chitosan. Plant Sci Lett 33: 221–230 [Google Scholar]
  29. Konno H, Yamaya T, Yamasaki Y, Matsumoto H (1984) Pectic polysaccharide breakdown of cell walls in cucumber roots grown with calcium starvation. Plant Physiol 76: 633–637 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kuo CG, Tsay JS, Tsai CL, Chen RJ (1981) Tipburn of Chinese cabbage in relation to calcium nutrition and distribution. Sci Hortic (Amsterdam) 14: 131–138 [Google Scholar]
  31. Leijon F, Melzer M, Zhou Q, Srivastava V, Bulone V (2018) Proteomic analysis of plasmodesmata from Populus cell suspension cultures in relation with callose biosynthesis. Front Plant Sci 9: 1681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Li B, Kamiya T, Kalmbach L, Yamagami M, Yamaguchi K, Shigenobu S, Sawa S, Danku JMC, Salt DE, Geldner N, et al. (2017) Role of LOTR1 in nutrient transport through organization of spatial distribution of root endodermal barriers. Curr Biol 27: 758–765 [DOI] [PubMed] [Google Scholar]
  33. Luna E, Pastor V, Robert J, Flors V, Mauch-Mani B, Ton J (2011) Callose deposition: A multifaceted plant defense response. Mol Plant Microbe Interact 24: 183–193 [DOI] [PubMed] [Google Scholar]
  34. Marschner H. (2011) Marschner’s Mineral Nutrition of Higher Plants. Academic Press, London [Google Scholar]
  35. Mur LA, Kenton P, Atzorn R, Miersch O, Wasternack C (2006) The outcomes of concentration-specific interactions between salicylate and jasmonate signaling include synergy, antagonism, and oxidative stress leading to cell death. Plant Physiol 140: 249–262 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nagasaki H, Mochizuki T, Kodama Y, Saruhashi S, Morizaki S, Sugawara H, Ohyanagi H, Kurata N, Okubo K, Takagi T, et al. (2013) DDBJ read annotation pipeline: A cloud computing-based pipeline for high-throughput analysis of next-generation sequencing data. DNA Res 20: 383–390 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nishimura MT, Stein M, Hou BH, Vogel JP, Edwards H, Somerville SC (2003) Loss of a callose synthase results in salicylic acid-dependent disease resistance. Science 301: 969–972 [DOI] [PubMed] [Google Scholar]
  38. Okada H, Abe M, Asakawa-Minemura M, Hirata A, Qadota H, Morishita K, Ohnuki S, Nogami S, Ohya Y (2010) Multiple functional domains of the yeast l,3-β-glucan synthase subunit Fks1p revealed by quantitative phenotypic analysis of temperature-sensitive mutants. Genetics 184: 1013–1024 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Olle M, Bender I (2009) Causes and control of calcium deficiency disorders in vegetables: A review. J Hortic Sci Biotechnol 84: 577–584 [Google Scholar]
  40. Powell DA, Morris ER, Gidley MJ, Rees DA (1982) Conformations and interactions of pectins. II. Influences of residue sequence on chain association in calcium pectate gels. J Mol Biol 155: 517–531 [DOI] [PubMed] [Google Scholar]
  41. Provart N, Zhu T (2003) A browser-based functional classification SuperViewer for Arabidopsis genomics. Curr Comput Mol Biol 2003: 271–272 [Google Scholar]
  42. R Core Team (2018) R: A language and environment for statistical computing. R Foundation for Statistical Computing. https://www.R-project.org/
  43. Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: A Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26: 139–140 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Robinson MD, Oshlack A (2010) A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol 11: R25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Saure MC. (1998) Causes of the tipburn disorder in leaves of vegetables. Sci Hortic (Amsterdam) 76: 131–147 [Google Scholar]
  46. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, et al. (2012) Fiji: An open-source platform for biological-image analysis. Nat Methods 9: 676–682 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schmitz-Eiberger M, Haefs R, Noga G (2002) Calcium deficiency: Influence on the antioxidative defense system in tomato plants. J Plant Physiol 159: 733–742 [Google Scholar]
  48. Shikanai Y, Yamagami M, Shigenobu S, Yamaguchi K, Kamiya T, Fujiwara T (2015) Arabidopsis thaliana PRL1 is involved in low-calcium tolerance. Soil Sci Plant Nutr 61: 951–956 [Google Scholar]
  49. Stein RJ, Höreth S, de Melo JRF, Syllwasschy L, Lee G, Garbin ML, Clemens S, Krämer U (2017) Relationships between soil and leaf mineral composition are element-specific, environment-dependent and geographically structured in the emerging model Arabidopsis halleri. New Phytol 213: 1274–1286 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Su T, Yu S, Yu R, Zhang F, Yu Y, Zhang D, Zhao X, Wang W (2016) Effects of endogenous salicylic acid during calcium deficiency-induced tipburn in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Plant Mol Biol Rep 34: 607–617 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Taylor MD, Locascio SJ (2004) Blossom-end rot: A calcium deficiency. J Plant Nutr 27: 123–139 [Google Scholar]
  52. Thiele K, Wanner G, Kindzierski V, Jürgens G, Mayer U, Pachl F, Assaad FF (2009) The timely deposition of callose is essential for cytokinesis in Arabidopsis. Plant J 58: 13–26 [DOI] [PubMed] [Google Scholar]
  53. Töller A, Brownfield L, Neu C, Twell D, Schulze-Lefert P (2008) Dual function of Arabidopsis glucan synthase-like genes GSL8 and GSL10 in male gametophyte development and plant growth. Plant J 54: 911–923 [DOI] [PubMed] [Google Scholar]
  54. Trapnell C, Pachter L, Salzberg SL (2009) TopHat: Discovering splice junctions with RNA-Seq. Bioinformatics 25: 1105–1111 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Truernit E, Bauby H, Dubreucq B, Grandjean O, Runions J, Barthélémy J, Palauqui JC (2008) High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of phloem development and structure in Arabidopsis. Plant Cell 20: 1494–1503 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Ueoka-Nakanishi H, Tsuchiya T, Sasaki M, Nakanishi Y, Cunningham KW, Maeshima M (2000) Functional expression of mung bean Ca2+/H+ antiporter in yeast and its intracellular localization in the hypocotyl and tobacco cells. Eur J Biochem 267: 3090–3098 [DOI] [PubMed] [Google Scholar]
  57. Walker RB, Walker HM, Ashworth PR (1955) Calcium-magnesium nutrition with special reference to serpentine soils. Plant Physiol 30: 214–221 [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. White PJ, Broadley MR (2003) Calcium in plants. Ann Bot 92: 487–511 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wildermuth MC, Dewdney J, Wu G, Ausubel FM (2001) Isochorismate synthase is required to synthesize salicylic acid for plant defence. Nature 414: 562–565 [DOI] [PubMed] [Google Scholar]
  60. Wu Q, Shigaki T, Han JS, Kim CK, Hirschi KD, Park S (2012) Ectopic expression of a maize calreticulin mitigates calcium deficiency-like disorders in sCAX1-expressing tobacco and tomato. Plant Mol Biol 80: 609–619 [DOI] [PubMed] [Google Scholar]
  61. Wu Z, Liang F, Hong B, Young JC, Sussman MR, Harper JF, Sze H (2002) An endoplasmic reticulum-bound Ca2+/Mn2+ pump, ECA1, supports plant growth and confers tolerance to Mn2+ stress. Plant Physiol 130: 128–137 [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Yamaguchi K, Takahashi Y, Berberich T, Imai A, Miyazaki A, Takahashi T, Michael A, Kusano T (2006) The polyamine spermine protects against high salt stress in Arabidopsis thaliana. FEBS Lett 580: 6783–6788 [DOI] [PubMed] [Google Scholar]

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