Genome-wide Identification and Expression Analysis of the YTH Domain-containing RNA-binding Protein Family in Citrus Sinensis

in Journal of the American Society for Horticultural Science

In eukaryotic systems, messenger RNA regulations, including splicing, 3′-end formation, editing, localization, and translation, are achieved by different RNA-binding proteins and noncoding RNAs. The YTH domain is a newly identified RNA-binding domain that was identified by comparing its sequence with that of splicing factor YT521-B. Previous study showed that the YTH gene plays an important role in plant resistance to abiotic and biotic stress. In this study, 211 YTH genes were identified in 26 species that represent four major plant lineages. Phylogenetic analysis revealed that these genes could be divided into eight subgroups. All of the YTH genes contain a YT521 domain and have different structures. Ten YTH genes were identified in navel orange (Citrus sinensis). The expression profiles of these CitYTH genes were analyzed in different tissues and at different fruit developmental stages, and CitYTH genes displayed distinct expression patterns under heat, cold, salt, and drought stress. Furthermore, expression of the CitYTH genes in response to exogenous hormones was measured. Nuclear localization was also confirmed for five of the proteins encoded by these genes after transient expression in Nicotiana benthamiana cells. This study provides valuable information on the role of CitYTHs in the signaling pathways involved in environmental stress responses in Citrus.

Contributor Notes

This work was financially supported by grants from Key R & D projects of Jiangxi (20171ACF60022), and Science and Technology Support Program of Jiangxi Province (20152ACF60007).

BZ and ZO designed the experiments. ZO, HD, LM, WH, and JC carried out most of the experiments. BZ, XL, and ZO drafted and revised the manuscript. All authors read and approved the final manuscript.

Corresponding authors. E-mail: or

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Article Figures

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    Species of plant investigated in this study. Left column: phylogenetic tree of plant species generated according to the classification of plants in the Phytozome database (Goodstein et al., 2011). Right column: YTH domain-containing proteins identified. A total of 211 YTH sequences were identified among 26 plant species.

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    Phylogenetic relationship between YTH domain-containing proteins (YTHs) identified from 26 plant species. YTH family members were clustered into three groups, and further divided into two, three, and three subgroups, respectively. Protein sequences of YTH domain-containing proteins were obtained from the Phytozome database (Goodstein et al., 2011). Multiple sequence alignments of the full-length YTH proteins from representative plant species were performed using the ClustalX program (Larkin et al., 2007). The phylogenetic tree was constructed using MEGA 6.0 by the neighbor-joining method (Tamura et al., 2013). Citrus sinensis YTH proteins are highlighted by the black circle.

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    Phylogenetic relationships, conserved motif compositions, numbers of introns, and gene structure of YTH domain-containing proteins. Left column: phylogenetic tree of YTH domain-containing proteins. Middle left column: exon/intron gene structure (filled box = exon, solid line = intron). The intron phases are indicated as the numbers 0, 1, and 2. Excluding DsYTH1, PvYTH11, SlYTH7, SlYTH8, SlYTH9, VvYTH2, VvYTH7, VvYTH9, and ZmYTH19, whose long introns are denoted by a “/”, the exons and introns are drawn to scale. Middle right column: numbers of introns. Right column: conserved domains within the YTH domain-containing proteins. The domains and motifs are drawn to scale.

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    Expression analysis of Citrus sinensis YTH domain-containing protein genes in the different tissues of ‘Gannan Zao’ navel orange plants (R = roots, S = stems, L = leaves, F = flowers, FR = mature fruit pulp, FP = mature fruit peels). Root, stem, and leaf samples were collected from spring shoots (≈2 months after grafting). Flowers at full bloom and whole fruits at 140 d after full bloom were collected from adult navel orange trees grown in a culture chamber. The transcript levels of CitYTH genes were detected using the real-time polymerase chain reaction. The data presented are mean ± sd of three independent experiments. The x- and y-axes represent the different tissues of navel orange and relative expression (fold change relative to actin transcript values), respectively.

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    Heatmap representation of Citrus sinensis YTH domain-containing protein genes in fruit peels and pulp during fruit development. Whole fruits at 140, 160, and 200 d after full bloom (DAFB) were collected from 4-year-old ‘Gannan Zao’ navel orange trees. Transcriptome sequencing (RNA-Seq) analysis was used to analyze the expression of CitYTH genes, and each experiment contains three biological replications. The heatmap was generated in R using the heatmap 2 function from the gplots CRAN library (Warnes et al., 2013). The bar on the right of heatmap represents the relative expression value. 1P_WT, 2P_WT, and 3P_WT = peels of ‘Newhall’ fruit at 160, 180, and 200 DAFB, respectively; 1P_MT, 2P_MT, and 3P_MT = peels of ‘Gannan Zao’ fruit at 160, 180, and 200 DAFB, respectively; 1R_WT, 2R_WT, and 3R_WT = pulp of ‘Newhall’ fruit at 160, 180, and 200 DAFB, respectively; 1R_WT, 2R_WT, and 3R_WT = pulp of ‘Gannan Zao’ fruit at 160, 180, and 200 DAFB, respectively.

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    Expression analysis of Citrus sinensis YTH domain-containing protein genes in ‘Gannan Zao’ navel orange leaves treated with drought or salt stress. Drought stress was applied to 3-week-old navel orange seedlings after grafting by transferred to three layers of filter papers for fast dehydration. Salt stress was applied to 3-week-old seedlings by drenching the plants with a 200-mm NaCl solution. Navel orange leaves were collected for quantitative polymerase chain reaction analysis at 0, 24, and 48 h after treatment. Relative expression folds were calculated after normalization with CitActin transcript values. The x- and y-axes represent the treatment time (units = hours) and relative expression, respectively. The data presented are mean ± sd of three independent experiments run in triplicate. The * above the columns indicates significant differences at the P < 0.05 level.

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    Expression analysis of Citrus sinensis YTH domain-containing protein genes in ‘Gannan Zao’ navel orange leaves treated with cold or heat stress. Cold and heat stress was applied by placing 3-week-old navel orange seedlings after grafting in growth chambers whose temperatures were set at 4 and 42 °C, respectively; navel orange trees grown at 28 °C were used as controls. Leaf samples for quantitative polymerase chain reaction analysis were collected at 0, 24, and 48 h after treatment. The x- and y-axes represent the treat time (units = hours) and relative expression (fold change relative to actin transcript values), respectively. The data presented are mean ± sd of three independent experiments run in triplicate. The * above the columns indicates significant differences at the P < 0.05 level.

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    Expression analysis of Citrus sinensis YTH domain-containing protein genes in ‘Gannan Zao’ navel orange leaves treated with salicylic acid (SA), methyl jasmonate (JA), and abscisic acid (ABA). Three-week-old navel orange seedlings after grafting were treated by foliar sprays of 1 mm SA, 100 μM JA, 100 μM ABA, or an equal volume of solvent [as a control (ck)]. The expression of CitYTH genes was analyzed by quantitative polymerase chain reaction after the seedlings were treated for 0, 24, and 48 h. The x- and y-axes represent the treat time (units = hours) and relative expression (fold change relative to actin transcript values), respectively. The data presented are mean ± sd of three independent experiments run in triplicate. The * above the columns indicates a significant difference at the P < 0.05 level.

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    Subcellular location of Citrus sinensis YTH domain-containing proteins. Nicotiana benthamiana leaves were transiently transformed with constructs harboring pFGC-Egfp:CitYTHs and pFGC-Egfp via agroinfiltration. Fluorescence of the target proteins was observed with confocal microscopy 36 h post infection.

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