Characterization of the Dioscorin Gene Family in Dioscorea alata Reveals a Role in Tuber Development and Environmental Response

Dioscorin is one of the major soluble proteins in yam tubers. Unlike other well-known plant storage proteins, such as patatin and sporamin, dioscorin is argued for its function as storage proteins, and the molecular mechanisms underlying its expressional complexity are little understood. In this study, we isolated five dioscorin genes from Dioscorea alata L., comprising three class A (Da-dio1, -3 and -4) and two class B (Da-dio2 and -5) isoforms. Expressions of all dioscorin genes gradually decreased in mother tubers during yam sprouting and regrowth. On the other hand, all dioscorin genes accumulated transcripts progressively with tuber development in new tubers, with Da-dio5 being the most prominent isoform. In yam leaves, the expressions of Da-dio5 were up-regulated by the treatments of five phytohormones (gibberellic acid, salicylic acid, indole-3-acetic acid, abscisic acid, and ethylene), and three abiotic stresses (high-temperature, low-temperature and drought). To further elucidate the regulatory mechanisms of Da-dio5 expressions, transgenic Arabidopsis plants harboring the Da-dio5 promoter-β-glucuronidase (GUS) fusion were generated. GUS staining showed that expressions of the Da-dio5 promoter were detected mainly in the shoot apical meristem (SAM) and hypocotyls, and enhanced by the treatments of the five hormones, and the three abiotic stresses mentioned above. These results suggest diverse roles of Da-dio5 in yam sprouting, regrowth, and tuberization, as well as in response to enviromental cues.


Introduction
Yams (Dioscorea spp.) are members of the monocotyledonous family Dioscoreaceae. More than 600 yam species have been cultivated worldwide [1], consumed as an important source of food in some African and Asian countries due to the high contents of carbohydrate and nutritionally relevant proteins (1-3%) in fresh tubers [2,3]. In China, yams are traditionally used as medicinal foods to strengthen the spleen, kidney, liver, and stomach; to reduce phlegm and fatigue; and to cure chronic diarrhea and diabetes [4]. The beneficial properties of yams have recently been attributed to the

Identification of the Da-dio Gene Family
The full-length cDNAs of five Da-dio genes were cloned and submitted to GeneBank (Da-dio1, KX237676; Da-dio2, KX237677; Da-dio3, KX237678; Da-dio4, KX237676; Da-dio5, KX237676). The open reading frame (ORF) of the five Da-dio genes were 819-831 base pair (bp) long, and predicted proteins of 272-276 amino acids (aa), molecular weights (MW) of~30 kDa, and theoretical isoelectric point (pI) values of 6.17 to 7.02 (Table 1). Multiple sequence alignments were performed upon the five Da-dio proteins identified here, a dioscorin homolog (dioscorin-5 precursor) from Dioscorea japonica and three carbonic anhydrases (CAs) from human (P00915), mouse (P13634) and Arabidopsis (CAB79100), respectively. As shown in Figure 1A, the five Da-dios contained three putative residues that influence CA activity, and two cysteine residues that relate to activities of TI, DHA reductase, and MDA reductase. In addition, all dioscorin proteins contained a putative signal peptide (amino acids [23][24][25]. A phylogenetic tree ( Figure 1B) was constructed for the five Da-dios and 18 NCBI-downloaded dioscorin sequences of five Dioscorea species (D. alata, D. japonica, D. pseudojaponica, D. batatas and D. cayenensis). The five Da-dios identified this study were grouped clearly into class A (Da-dio1, Da-dio3 and Da-dio4) and class B (Da-dio2 and Da-dio5). Amino acid sequence alignments showed a high similarity among the Da-dios of different cultivars, being 91-97% identical within the same subfamily, and 68% similar across the subfamilies (Table S1 and Figure 1B).

Expression of the Five Da-dios During Tuber Development
D. alata L. propagates mainly via tubers, the modified stem. Tuber development is accompanied by a variety of biochemical and morphological changes. To explore the roles of the five Da-dio genes during yam sprouting and regrowth, we examined their expressions in the mother tubers at 15, 30, 45, 60, 75 and 90 days after planting (DAP). All five Da-dio genes were expressed at highest abundance in mother tubers at 15 DAP. As the vines continued to grow, the expression of the five Da-dio genes gradually decreased until reaching almost undetectable levels at 90 DAP ( Figure 2A). To further investigate whether the dioscorin gene expressions are associated with tuber development, the Da-dio transcripts were investigated in new tubers at 120, 150, 180, 210 and 240 DAP, corresponding to different stages of tuber development ( Figure 2B). As shown in Figure 2C, the expressions of five Da-dio genes increased in a similar pattern in the new tubers except for a difference in the fold-change. The expressions of Da-dio1-4 were up-regulated by 3-5-fold, whereas almost 300-fold was observed for Da-dio5 at the beginning of the maturation stage (210 DAP). These results suggested that expressions of the five dioscorin genes are closely correlated with yam sprouting, regrowth, and tuber development, of which Da-dio5 seems to be the most important member implicated in tuber formation and development.
for Da-dio5 at the beginning of the maturation stage (210 DAP). These results suggested that expressions of the five dioscorin genes are closely correlated with yam sprouting, regrowth, and tuber development, of which Da-dio5 seems to be the most important member implicated in tuber formation and development. Values are presented as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences (p < 0.05) according to one-way analysis of variance.

Expression Pattern and Localization of Da-dio5
To further understand the functions of Da-dio5, its expressions were examined in six D. alata tissues, viz. tubers, stems, roots, bulbils, male flowers and leaves. Da-dio5 exhibited a tissue-specific expression, with transcripts detected mainly in tubers and bulbils but hardly in the other tissues ( Figure 2D), further strengthening the importance of Da-dio5 in yam tuber development. To identify the cellular compartment in which Da-dio5 functions, the Da-dio5 protein fused with a green fluorescent protein (GFP) was transiently expressed in rice protoplasts. As shown in Figure 3, Da-dio5-GFP was localized to the vacuole and cytosol of transformed protoplasts.

Expression of Da-dio5 in Response to Hormones and Abiotic Stresses
To further explore the expressional characters of Da-dio5, leaves of young yam plants were treated with five hormones (GA, gibberellic acid; SA, salicylic acid; IAA, indole-3-acetic acid; ABA, abscisic acid; ET, ethylene.) and three stresses (drought, 4 °C and 45 °C). As shown in Figure 4A, expressions of the Da-dio5 in leaves were enhanced at early stages of the hormone treatments (3-6 h), Values are presented as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences (p < 0.05) according to one-way analysis of variance.

Expression Pattern and Localization of Da-dio5
To further understand the functions of Da-dio5, its expressions were examined in six D. alata tissues, viz. tubers, stems, roots, bulbils, male flowers and leaves. Da-dio5 exhibited a tissue-specific expression, with transcripts detected mainly in tubers and bulbils but hardly in the other tissues ( Figure 2D), further strengthening the importance of Da-dio5 in yam tuber development. To identify the cellular compartment in which Da-dio5 functions, the Da-dio5 protein fused with a green fluorescent protein (GFP) was transiently expressed in rice protoplasts. As shown in Figure 3, Da-dio5-GFP was localized to the vacuole and cytosol of transformed protoplasts.

Expression of Da-dio5 in Response to Hormones and Abiotic Stresses
To further explore the expressional characters of Da-dio5, leaves of young yam plants were treated with five hormones (GA, gibberellic acid; SA, salicylic acid; IAA, indole-3-acetic acid; ABA, abscisic acid; ET, ethylene.) and three stresses (drought, 4 • C and 45 • C). As shown in Figure 4A, expressions of the Da-dio5 in leaves were enhanced at early stages of the hormone treatments (3-6 h), but depressed in different degrees for respective hormones at later stages. The expressions of Da-dio5 in leaves were significantly up-regulated by the drought treatment, and the two temperature stresses, although to a much lesser extent ( Figure 4B). but depressed in different degrees for respective hormones at later stages. The expressions of Da-dio5 in leaves were significantly up-regulated by the drought treatment, and the two temperature stresses, although to a much lesser extent ( Figure 4B).   but depressed in different degrees for respective hormones at later stages. The expressions of Da-dio5 in leaves were significantly up-regulated by the drought treatment, and the two temperature stresses, although to a much lesser extent ( Figure 4B).

Functional Characterization of the Da-dio5 Promoter
To characterize the spatiotemporal expression patterns of Da-dio5, a 2617-bp putative promoter fragment upstream of Da-dio5 was cloned using a PCR-based genome walking technique. Multiple types of putative cis-acting elements were identified in the Da-dio5 promoter ( Table 2). The Da-dio5 promoter contained a GCN4_motif and a Skn-1_motif, which are associated with endosperm expression. The promoter also harbored putative regulatory elements responsible for responses to environmental stresses and hormone signals, such as low-temperature and heat-stress responsive elements, drought-inducible elements, defense-responsive elements, and elements responsive to various hormones (ET, SA, GA, ABA, and IAA). For a precise analysis of the expression patterns of the Da-dio5 promoter, we transformed the Da-dio5 promoter::GUS chimeric construct into A. thaliana. In transgenic Arabidopsis seedlings at 10 days after germination (DAG), GUS staining was predominantly observed in the shoot apical meristem (SAM) and hypocotyls, while the petiole was weakly stained ( Figure 5A). In transgenic seedlings at 20 DAG, a similar staining pattern was observed in the SAM, petiole and vasculature, whereas no staining was observed in the hypocotyls. As expected, the blue stain was not observed in control seedlings. There was also a lack of staining in the roots, flowers and stems at any developmental stages (not shown), indicating the tissue/organ-specific activity of the Da-dio5 promoter. To determine whether the cis-acting elements as predicted in hormone and stress responses (Table 2) were functional, histochemical GUS staining and GUS activity assay were conducted in transgenic seedlings. GUS activity was significantly induced in seedlings at 9 h after the treatments of GA, IAA, SA, or ABA. In contrast, in ACC-treated seedlings, GUS gene expression increased at 6 h, and then decreased at 9 h ( Figure 5B). GUS activity was enhanced by both high-temperature and drought treatments, with the latter being more striking, but inhibited markedly by low-temperature treatment ( Figure 5C). The GUS staining observations correlated well the assays of GUS activity ( Figure 5). These data were consistent with some of the Da-dio5 expression patterns ( Figure 4) and clearly indicated that the Da-dio5 has crucial functions in responding to various hormone and environmental cues. 20-day-old transgenic seedlings. Bar = 1 mm for (a) and 1 cm for (b); (B) GUS activity analysis in 10-day-old transgenic seedlings after GA, SA, IAA, ABA, and ACC treatments. Bar = 1 mm; (C) GUS activity analysis in transgenic seedlings after low-temperature (4 °C), high-temperature (45 °C), and drought treatments. Ten-day-old transgenic seedlings were used for 4 °C and 45 °C treatments, bar = 1 mm; 20-day-old transgenic seedlings were used for drought treatment, bar = 1 cm. Values are presented as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences (p < 0.05) according to one-way analysis of variance.

Discussion
Dioscorin, which is the major soluble protein in yam (Dioscorea spp.) tubers, has been isolated from various yam species [12]. In previous reports, phylogenetic analysis of dioscorins from various yam species revealed that the dioscorins were grouped into two classes (A and B) [12]. Here, we showed the presence of five dioscorin genes in D. alata cv. Hainan No. 56 that consists of three class A (Da-dio1, -3 and -4) and two class B (Da-dio2 and -5) isoforms. Phylogenetically, the five dioscorin genes are highly conserved, similar to their homologs in other Dioscorea species, implying the subjection of these genes to a similar selection pressure during evolution. Moreover, complete conservation of two cysteine residues that relate to activities of TI, DHA reductase, and MDA reductase suggest additional roles besides functions as storage proteins. Ten-day-old transgenic seedlings were used for 4 • C and 45 • C treatments, bar = 1 mm; 20-day-old transgenic seedlings were used for drought treatment, bar = 1 cm. Values are presented as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences (p < 0.05) according to one-way analysis of variance.

Discussion
Dioscorin, which is the major soluble protein in yam (Dioscorea spp.) tubers, has been isolated from various yam species [12]. In previous reports, phylogenetic analysis of dioscorins from various yam species revealed that the dioscorins were grouped into two classes (A and B) [12]. Here, we showed the presence of five dioscorin genes in D. alata cv. Hainan No. 56 that consists of three class A (Da-dio1, -3 and -4) and two class B (Da-dio2 and -5) isoforms. Phylogenetically, the five dioscorin genes are highly conserved, similar to their homologs in other Dioscorea species, implying the subjection of these genes to a similar selection pressure during evolution. Moreover, complete conservation of two cysteine residues that relate to activities of TI, DHA reductase, and MDA reductase suggest additional roles besides functions as storage proteins.
Tuber development includes three major steps: induction, initiation and tuberization. Each of these steps is accompanied by a drastic change in the expression of genes encoding a set of proteins abundantly present in tubers [21]. However, it is unclear which genes are required for tuber development. In contrast to the established roles of potato (patatin) and sweet potato (sporamin) storage proteins in tuber development [21][22][23][24][25][26], dioscorins have been studied mainly for their biological activities and pharmaceutical potentials [5]. In this study, five dioscorin genes exhibited similar expression patterns during yam sprouting, regrowth and tuberization (Figure 2). The dioscorin gene expression levels gradually decreased until they were undetectable with the process of tuber germination, sprouting and regrowth (Figure 2A). Tuber growth is reported to be accompanied by protein (dioscorin) depletion along with sugar mobilization, which is controlled by the redox status of the tubers [27]. Increasing evidences have shown that dioscorins possess CA, DHA reductase and MDA reductase activities that are important for control of redox status in D. alata L. tubers [28]. Thus, these results together conclude that dioscorins function as the major storage protein to support tuber germination, and provide nutrients for the growth of new plants from reproductive organs. Additionally, all five dioscorin isoforms were detected at 120 DAP in accordance with tuber formation, and were highly abundant in maturing tubers (at 210 days), with Da-dio5 being the most prominent isoform ( Figure 2C). Previous studies showed that the process of tuberization is accompanied by increased oxidative stress, evidenced by elevated levels of H 2 O 2 and reactive nitrogen species. Therefore, it is suggested dioscorins have an role in controlling redox status that is also important for the process of tuberizaion [28]. Further study revealed the expressional predominance of Da-dio5 in the types of reproductive organs: tubers and bulbils ( Figure 2D). Together, these results suggest the importance of dioscorins in both vegetative growth and reproductive development in yam plants.
The findings in sweet potato illustrate the precise roles of the phytohormone and signaling pathways in regulating expressions of tuber storage proteins (sporamin) upon abiotic stresses [29]. In this study, the expressions of Da-dio5 in yam leaves were up-regulated by the treatments of five phytohormones (GA, SA, IAA, ABA, and ET), and three abiotic stresses (high-temperature, low-temperature and drought) ( Figure 4A,B). Thus, the induced expression of Da-dio5 revealed its involvement in adaptation to changes of environmental cues and stresses. Furthermore, under field conditions, induction of tuberization is controlled by a number of environmental conditions such as photo-period, temperature and nitrogen supply, via the change of endogenous phytohormone levels. Considering the expressional predominance of Da-dio5 in yam tubers, and the coincidence of increased dioscorin expression with yam tuberization, characterization of the Da-dio5 promoter will be beneficial to understanding the regulatory mechanisms of dioscorin genes in relation to their physiological functions in yam plants. Transgenic Arabidopsis seedlings harboring the Da-dio5 promoter-GUS construct showed GUS staining mainly in the SAM and hypocotyls at 10 DAG, but absent in the hypocotyls at 20 DAG ( Figure 5A), indicating a strict spatial/temporal expression pattern. This is consistent with the findings that the underground stem tubers of yam are derived from swollen hypocotyls [1]. In addition, both GUS activity assay and GUS staining showed that the Da-dio5 promoter was also induced by multiple phytohormones (GA, SA, IAA, ABA, and ACC), high-temperature and drought treatments, but inhibited by low-temperature treatment ( Figure 5B,C), suggesting a multifaceted response of Da-dio5 to changeable environmental cues. These responses could be backed up by the relevant cis-elements predicted in the Da-dio5 promoter ( Table 2), suggesting our observations were similar to those of tuber storage protein homologs in potato and sweet potato [30,31].

Isolation of Dioscorin Genes
A D. alata cv. Hainan No. 56 transcriptome database that includes a total of 52,866 unigenes was obtained by RNA-sequencing and de novo assembly using the Illumina HiSeq 2000 platform (data, unpublished). The sequences of dioscorin genes from other yam species were downloaded from the National Center for Biotechnology Information (NCBI) database and used as query sequences for basic local alignment search tool (BLAST) analysis of our transcriptome database. We obtained five D. alata dioscorin (Da-dio) target sequences with complete open reading frames. Subsequently, specific primer pairs were designed to amplify respective Da-dio cDNA sequences (Table S2).

Multiple Sequence Alignments and Bioinformatic Analysis
We deposited the obtained sequences to the NCBI database for basic local alignment search tool (BLAST) searches, following the online comparative and bioinformatic analyses of the Da-dio genes (Available online: http://www.ncbi.nlm.nih.gov). The amino acid sequences of the five Da-dios were aligned with homologous sequences downloaded from NCBI using the DNAMAN (Lynnon Biosoft, Quebec, QC, Canada) program. Additionally, the neighbor-joining method of the MEGA6 program was used to construct a phylogenetic tree. The molecular weights and theoretical pI (isoelectric point) values of the dioscorins were calculated with the ProtParam online tool (Available online: http://www.expasy.ch/tools/protparam.html). Potential conserved domains and signal peptides were analyzed using the SMART program (Available online: http://smart.embl-heidelberg.de/).

RNA Isolation and cDNA Synthesis
Total RNA was isolated from frozen plant samples using the RNAplant Plus reagent (BioTeke, Beijing, China). First-strand cDNA was synthesized using the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Burlington, ON, Canada).

qRT-PCR
qRT-PCR assay was used to analyze Da-dio gene expressions, with β-actin and α-tubulin-1 as internal controls for data normalization. The efficiency of each primer pair was determined to be 1.946-2.001. The qRT-PCR was conducted using SYBR ® Premix Ex Taq™ II (Takara, Dalian, China) and the LightCycler 2.0 system (Roche Diagnostics, Basel, Switzerland). We used the LightCycler Relative Quantification Software 4.05 (Roche Diagnostics, Mannheim, Germany) to visualize and analyze the data, including the quantification cycle values, PCR efficiencies, and correlation coefficients. We calculated relative expression level fold changes using previously described methods [37].

Subcelluar Localization of Da-dio5
For subcellular localization analysis, the Da-dio5 cDNA was amplified using the specific primer pairs of Da-dio5-GF and Da-dio5-GR (Table S3), and then cloned into the binary vector pCAMBIA-1300 at the restriction sites of Sal I and Kpn I to make the 35S::Da-dio5-GFP fusion construct. Fusion construct was transformed into Oryza sativa mesophyll protoplasts according to a previous study [38]. The images were acquired with laser scanning confocal microscope (Olympus FV1000, Tokyo, Japan). The excitation and emission wavelengths were 480 nm and 510 nm for GFP, and 650 nm and 750 nm for chloroplast, respectively.

Promoter Cloning and Analysis
The 5 -flanking region of the Da-dio5 gene was isolated using the GenomeWalker Universal Kit (Takara, Dalian, China). The GenomeWalker libraries were constructed using genomic DNA extracted from yam leaves using the cetyltrimethylammonium bromide extraction method as previously described [39]. Primary and nested PCRs were conducted using adapter primers provided by the kit and three gene-specific antisense primers (Table S3). The PCR products were ligated into the pMD18-T vector (Takara, Dalian, China), and then sequenced at Invitrogen, China. Regulatory elements in the promoter were analyzed using the Plantcare online program (Available online: http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [40].

Construction of the Da-dio5 Promoter-GUS Fusion and A. thaliana Transformation
To construct the Da-dio5 promoter-GUS fusion, the 1500-bp Da-dio5 promoter fragment was subcloned into the Sac I-Nco I site of pCAMBIA1301. The construct was transferred into Agrobacterium tumefaciens EHA105 cells using the liquid nitrogen freeze-thaw method [35] for the subsequent transformation of Arabidopsis plants using the floral dip method [41]. Homozygous plants were selected from the T 2 progenies and confirmed in the T 3 generation according to hygromycin (50 µg/mL) resistance.

Histochemical Localization and Quantitative Analysis of GUS Activity
Histochemical localization of GUS activity in the homozygous transgenic plants was conducted as described [36]. Quantitative analysis of GUS activity in transgenic seedlings was as described by Lü et al. [41]. Each assay was repeated three times, with three technical replicates each time.