Search for Candidate Genes Causing the Excessive Ca Accumulation in Roots of Tipburn-Damaged Lisianthus (Eustoma grandiflorum) Cultivars

Occurrence of tipburn is a severe problem in the production of lisianthus cultivars. Previous studies have shown excessive Ca accumulation in the roots of tipburn-damaged cultivars, where the distribution of Ca to the tips of the top leaves is inhibited. However, few studies have investigated the association between Ca accumulation and gene expression in horticultural crops. To provide a list of candidate target genes that might be causing the excessive Ca accumulation in roots, we focused Ca2+ transporter and pectin methylesterase (PME) genes and RNA-seq of upper leaves and roots in tipburn-occurrence cultivar (“Voyage peach”: VP) and non-occurrence cultivar (“Umi honoka”: UH) was conducted. In both the upper leaves and roots of VP, genes encoding the glutamate receptors (GLRs), cation/Ca2+ exchangers 4 (CCX4), Na+/Ca2+ exchanger-like protein (NCL), and PMEs were upregulated, and a gene encoding the cyclic nucleotide-gated ion channel 9 (CNGC9) was downregulated. In contrast, genes encoding the vacuolar cation/proton exchanger 5 (CAX5), calcium-transporting ATPase 1 and 12 (ACA1 and ACA12) showed differential expression in each organ. Among them, only CAX5 was upregulated and ACA12 was downregulated in the roots of VP. Based on these results, we suggested that CAX5 and ACA12 are the candidate genes causing the excessive Ca accumulation in the roots of tipburn-occurrence lisianthus cultivars. Future studies should investigate the temporal changes in gene expression using quantitative PCR and conduct functional analysis of candidate genes in tipburn-damaged lisianthus cultivars.


Introduction
Calcium (Ca) is an essential plant macronutrient and plays a vital role in plant growth. It acts as a counter cation in storage organelles and a crucial intracellular second messenger that provides protection against stresses [1]. The concentration of Ca 2+ in the cytosol (cytosol [Ca 2+ ]) is tightly regulated by three classes of transporters: Ca channels, Ca 2+ -ATPases (pumps), and Ca 2+ /cation antiporters [2,3]. To maintain cellular Ca 2+ homeostasis, these transporters have a pivotal role.
In horticultural crop production, enough Ca is generally supplied in the field. However, the occurrence of Ca deficiency disorders is observed in certain crops (e.g., tomato, lettuce, and Chinese cabbage) and causes serious economic losses [4][5][6]. It is suggested that Ca deficiency disorders are caused by the inability of the plant to translocate adequate Ca to the symptomatic organs, not the inability to acquire enough Ca [7][8][9]. In lisianthus (Eustoma grandiflorum (Raf.) Shinn.) cultivars, occurrence of tipburn (Ca deficiency disorder in the tips of new leaves) is a major problem in their production. Lisianthus is native to warm regions of the Southern United States and Northern Mexico. Its cultivars are mainly supplied as cut flowers. Previous research on occurrence of tipburn in lisinathus has suggested that differences in tipburn incidence and severity among the cultivars is af-Agriculture 2021, 11, 254 2 of 10 fected little by Ca acquisition, plant growth rate, and transpiration rate [10,11]. In contrast, excessive Ca accumulation in roots under a high Ca supply was observed in tipburndamaged cultivars [12,13]. Moreover, cultivars in which tipburn occurred had increased Ca distribution to the roots before and after the onset of tipburn, and inhibited Ca distribution to the leaves [11]. Thus, it is clear that Ca accumulation in the root is a key factor in the incidence of tipburn in lisianthus cultivars. As far as we know, the clear phenotypic response (i.e., excessive Ca accumulation in roots) was not observed in other crops although several studies have been conducted to find the causative genes of tipburn using transcriptome analysis and QTL analysis [14][15][16]. Therefore, the search for candidate genes causing the excessive Ca accumulation in roots of tipburn-damaged lisianthus cultivars must provide new insights to identify the causative genes of tipburn.
In plant physiology, the relevance of cellular Ca 2+ accumulation and the three classes of transporters has been investigated. Conn et al. [17] revealed that CAX1 (Ca 2+ /H + antiporter), ACA4, and ACA11 (Ca 2+ -ATPases) were preferentially expressed in the Carich mesophyll of Arabidopsis thaliana leaves. Further, their analysis of loss-of-function mutants demonstrated that CAX1 is a key regulator of the concentration of Ca 2+ in the apoplast (apoplastic [Ca 2+ ]) through compartmentalization of Ca 2+ into mesophyll vacuoles. Transgenic tomatoes [18,19] and potatoes [20,21], expressing sCAX1 from A. thaliana, increased total Ca content and caused a Ca deficiency disorder because they accumulated Ca 2+ in vacuoles and their apoplastic [Ca 2+ ] reduced.
As well as vacuoles, the plant cell wall is also a big pool of Ca 2+ in plant tissue. Ca 2+ in the cell wall has a structural role, crosslinking with the homogalacturonan (HG) domain of pectin. Pectin methylesterases (PMEs) are key factors regulating the binding of the HG domain to Ca 2+ . PMEs dimethyl esterify certain glycan regions within the HG domain, enabling Ca 2+ crosslink formation [22]. PME-silenced tomatoes exhibited lower blossom-end rot (Ca deficiency in fruit) incidence and higher apoplastic [Ca 2+ ] than those of wild-type [23]. Therefore, excessive Ca accumulation in tipburn-damaged lisianthus cultivars may be caused by the overexpression of genes encoding the three classes of transporters and PMEs.

Plant Materials
"Voyage peach" (VP) (Sakata Seed Corporation, Yokohama, Japan) and "Umi honoka" (UH) (Sumika Agrotech Co., Ltd., Osaka, Japan) cultivars were selected as a tipburndamaged cultivar and a tipburn-absent cultivar, respectively [13]. Before the onset of tipburn, VP exhibited a higher Ca concentration in each organ (whole leaves, stems, and roots) than was seen in UH (Table 1). After the onset of tipburn, whole leaf Ca concentrations in VP were lower than those of UH although VP had more than twice the Ca accumulation in the roots than UH (Table 1). Please see a previous study for more detail [13]. Table 1. Ca concentrations in each organ, tipburn incidence and severity of tipburn-absent cultivar (UH) and tipburndamaged cultivar (VP) at weeks 4 and 8 cited from a previous study [13].

Cultivars
Ca Treatments For the Ca concentrations of each organ, ANOVA was conducted to assess the effects of the treatment in each cultivar and significant differences among the means are indicated with different letters. n.s. represents no significant differences among the treatments. Cultivars: Umi honoka (UH) and Voyage peach (VP).
Seedlings were grown in the same way as in previous studies [10][11][12][13]. After plugs were transplanted into 0.25 L polyethylene pots, plants were supplied with a nutrient solution by bottom watering for 30 min once a day. The nutrient solution contained a 80 ppm Ca concentration, which was made by dissolving nutrient salts in distilled water 0.  [12,13].
Five weeks later, three tipburn-absent pots were randomly sampled from each cultivar. Harvested plants were washed with distilled water and divided into roots and top leaves for total RNA extraction.

RNA Extraction and RNA-Seq
For the three biological replications of leaves and roots in each cultivar, total RNA was extracted using NucleoSpin ® RNA Plant (Takara Bio Inc., Shiga, Japan). Contaminant DNA was eliminated using DNase and a quality check of RNA was conducted by Agilient Technologies 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). RNA Integrity Number (RIN) of all samples was greater than 8.0 and passed quality checks. TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, Inc., San Diego, CA, USA) was used for library preparation. The 12 libraries were sequenced using the NovaSeq 6000 (Illumina, Inc., San Diego, CA, USA). The read data were submitted to the DDBJ Read Archive (BioProject: PRJDB10656, BioSample: SAMD00252781-SAMD00252792).

Bioinformatics Analysis
Low reads, adaptor sequences, contaminant DNA, and PCR duplicates were removed from raw reads to reduce bias in the analysis. The clean reads were assembled de novo using Trinity with default parameters. For assembled genes, the longest contigs were filtered and clustered into the non-redundant transcripts using the CD-HIT-EST program. These transcripts were defined as "unigenes" and aligned to the assembled reference using Bowtie program. The assembled unigenes were annotated using BLASTX of DIAMOND with an E-value cut-off 1.0 × 10 −5 in the gene ontology (GO) and UniProt databases.
For the top leaves and roots, differentially expressed genes (DEGs) were contrasted within each cultivar (VP (tipburn-damaged cultivar) vs. UH (tipburn-absent cultivar)) using a threshold of p < 0.05. MA plots were drawn using DESeq2. For the target genes in this study, DEG lists, including genes with had log 2 fold-change values (log 2 FC) higher than 2 (upregulated genes) and less than −2 (downregulated genes), were created.

De Novo Transcriptome Assembly
A total of 234,725 transcripts and 160,301 genes were assembled from 12 cDNA libraries using clean reads (Table 2). Average contig length was 750.74 bp. All Q30 levels of each cultivar were higher than 96% (Table S1). Mapping ratios in each cultivar, obtained after aligning to the assembled reference were higher than 55% (Table S1).

Functional Annotation
For functional annotation of unigenes, the gene ontology (GO) database was applied to classify the annotated unigenes using BLASTX of DIAMOND with an E-value cut-off 1.0 × 10 −5 ( Figure S1). In the biological process category, the largest group was "metabolic process (35.04%)" ( Figure S1). In the cellular component category, the largest number of genes was mapped to the term "call part (46.92%)" ( Figure S1). In the molecular function category, "catalytic activity (46.47%)" was the largest group ( Figure S1).
To determine the expression of genes encoding the Ca channels (TPC1, GLRs, and CNGCs), Ca 2+ -ATPases (ECAs and ACAs), Ca 2+ /cation antiporters (CAXs, NCLs, and CCXs), and PMEs, we carried out BLASTX of DIAMOND with an E-value cut-off 1.0 × 10 −5 on UniProt database. The results for all cultivars and organs are summarized in Table S2. For the Ca channels, TPC1 was not detected while both GLRs and CNGCs were annotated with 13 genes (Table S2). For the Ca 2+ -ATPases, four ECAs and six ACAs were annotated (Table S2). For the Ca 2+ /cation antiporters, four CAXs, two NCLs, and three CCXs were annotated (Table S2). PMEs were annotated with 35 genes (Table S2).

Differentially Expressed Genes (DEGs)
For each organ, DEGs were contrasted between cultivars (VP (tipburn-damaged cultivar) vs. UH (tipburn-absent cultivar)) using the p-value threshold of < 0.05. MA plots were drawn using DESeq2 (Figure 1). In the upper leaves, 2240 contigs were upregulated and 1730 were downregulated in VP (the tipburn-damaged cultivar). In the roots, 1703 contigs were upregulated and 1673 were downregulated in VP.

Discussion
Before the onset of tipburn (4 w after the start of the experiment), VP exhibited higher Ca concentrations in each organ (whole leaves, stems, and roots) than those of UH (Table 1) [13]. In contrast, after the onset of tipburn (8 w after the start of the experiment), whole leaf Ca concentrations in VP were lower than those of UH although VP had more than twice the Ca accumulation in the roots than UH (Table 1) [13]. For each organ, DEGs were extracted as candidate genes related to excessive Ca accumulation just before the onset of tipburn (5 w after the start of the experiment) (Tables 3 and 4). From these results, a hypothetical diagram of cellular Ca dynamics in each organ was drawn in Figure 2.

Discussion
Before the onset of tipburn (4 w after the start of the experiment), VP exhibited higher Ca concentrations in each organ (whole leaves, stems, and roots) than those of UH (Table  1) [13]. In contrast, after the onset of tipburn (8 w after the start of the experiment), whole leaf Ca concentrations in VP were lower than those of UH although VP had more than twice the Ca accumulation in the roots than UH (Table 1) [13]. For each organ, DEGs were extracted as candidate genes related to excessive Ca accumulation just before the onset of tipburn (5 w after the start of the experiment) (Tables 3 and 4). From these results, a hypothetical diagram of cellular Ca dynamics in each organ was drawn in Figure 2.

Candidate Genes Causing the Difference of Ca Accumulation in Cultivars
In both upper leaves and roots, GLRs, CCX4, NCL, and PMEs were upregulated and CNGC9 were downregulated in VP (Tables 3 and 4 and Figure 2). These are the suggested candidate genes causing the difference in Ca accumulation in the cultivars before the onset Agriculture 2021, 11, 254 7 of 10 of tipburn (i.e., causing the higher Ca accumulation in each organ of VP rather than in UH 4 w after the start of the experiment [13]).
Glutamate-gated receptors (GLRs) are known to act as non-selective cation channels and the Arabidopsis genome contains a family of 20 GLR genes. Qi et al. [31] revealed that GLR3.3 plays a role in Ca 2+ influx channels at the plasma membrane, triggered directly or indirectly by six amino acids. In addition, Kim et al. [32] demonstrated that the overexpression of GLR3.2 leads to Ca 2+ deficiency symptoms by impairing the efficiency of Ca 2+ utilization. In the current study, most GLRs were upregulated in VP and more Ca accumulated than in UH. GLRs in lisianthus cultivars may have a relationship with Ca accumulation.
CCX4 has been identified as CAX10, but the results of the phylogenetic analysis showed that CCX4 is more closely related to the K + -dependent Na + /Ca 2+ antiporter than to any of the CAXs. In addition, AtCCX4-expressing cells can suppress the Na + and K + sensitivities of mutant yeast strains defective in vacuolar Na + and K + transport [33]. However, Corso et al. [34] demonstrated that AtCCX2 plays a key role in controlling the Ca 2+ fluxes between the endoplasmic reticulum (ER) and cytosol. Few functional analyses of CCX4 under high Ca concentrations have been conducted. Thus, effects of CCX4 expression on Ca accumulation in lisianthus should be investigated in more detail.
AtNCL (Na + /Ca 2+ exchanger-like protein) was identified as localizing in the Arabidopsis cell membrane fraction and plays an important role in Ca 2+ homeostasis under salt stress conditions [35]. In addition, AtNCL has the EF-hand Ca 2+ binding domain, and yeast cells expressing AtNCL accumulated more Ca 2+ than the wild-type under 30 mM CaCl 2 [36]. In the current experiment, NCL may contribute to the accumulation of more Ca in VP because the nutrient solution in this experiment was dissolved in more CaCl 2 and no NaCl.
Based on the results of a previous study [23], more Ca accumulation in cell walls and a decrease in apoplastic [Ca 2+ ] may have occurred in the VP because many PMEs were upregulated in their upper leaves and roots.
Tan et al. [37] demonstrated that CNGC9 is a Ca 2+ -permeable channel essential for constitutive root hair growth in Arabidopsis. However, few studies have investigated the relevance of CNGC9 expression to Ca accumulation. Effects of CNGC9 expression on Ca accumulation in lisianthus also should be investigated in more detail.

Candidate Genes Causing Excessive Ca Accumulation in Roots of VP
In contrast to the abovementioned genes, CAX5, ACA1, ACA12 showed different gene expression in each organ. CAX5 expression was not significantly different among the cultivars in the upper leaves while it was upregulated in the roots of VP. CAX5 is the Ca 2+ and Mn 2+ /H + antiporter in the vacuole and has autoinhibitory domains regulating Ca 2+ transport activity [38]. In addition, AtCAX5-transformed yeast showed higher Ca concentrations in the intracellular matrix than the control group under the same exposure to an electromagnetic field (EMF) [39]. Accordingly, excessive Ca accumulation in the roots of VP may be caused by excessive Ca accumulation in the vacuole through the overexpression CAX5.
Expressions of ACA1 in each organ were opposite to those of CAX5 (in the upper leaves, ACA1 was upregulated in VP; in roots, there was no significant difference in ACA1 expression among the cultivars). Huang et al. [40] demonstrated that ACA1 is a P-type Ca 2+ -ATPase localized in the inner plastid envelope of Arabidopsis. Its function is to maintain cytoplasmic Ca 2+ at micromolar concentrations [2,17]. In roots, increments of CAX5 expression and Ca accumulation in the vacuole may have resulted in decrements of ACA1 expression and Ca influx to the plastid (Figure 2).
ACA12 was upregulated in the upper leaves and downregulated in the roots of VP. ACA12 is localized in the plasma membrane and, unlike other ACAs, its activity is not stimulated by calmodulin [41]. Thus, ACA12 (calcium pump) activity is primarily regulated by increasing or decreasing mRNA expression [42]. Therefore, it is suggested that CAX5 and ACA12 are candidate genes causing the excessive Ca accumulation in roots of tipburn-damaged lisianthus cultivars. However, our experiment was only conducted just before the onset of tipburn (5 w after the start of the experiment). In addition, the threshold of p-value < 0.05 to be defined as DEGs may have been low (Figure 1). We need to comprehensively investigate the temporal changes in expression levels of annotated all genes (Table S2) using a quantitative PCR in the future. Functional analysis of candidate genes also should be conducted.

Conclusions
This study is the first attempt to provide a list of candidate target genes that might be causing the excessive Ca accumulation in roots of tipburn-damaged lisianthus cultivars. As a result, only two candidate genes were extracted. CAX5 was upregulated and ACA12 was downregulated in roots of a tipburn-damaged cultivar (VP). Therefore, tipburn-damaged cultivars may accumulate excessive Ca in the vacuole of roots through the overexpression of CAX5, inhibiting the distribution of Ca to the upper leaves. Further research including functional analysis of these candidate genes should be conducted.