Introduction

X1-homologous genes (XHS) encode plant specific proteins containing three basic domains XH, XS, and zf-XS which were named after Arabidopsis SGS3 (Suppessor of Gene Silencing 3) and rice homolog X1 (Mourrain et al. 2000; Bateman 2002; Qin et al. 2009). Among these protein domains, zf-XS motif is a C2H2 type zinc finger domain, the XS motif is an RNA-binding domain and XH motif refers to X-homolog domain with unknown function (Bateman 2002). Furthermore, these protein domains, some of SGS3-like proteins also include a coil–coil domain localized between the XS and XH domains (Ausin et al. 2009; Zheng et al. 2010).

The main region of rice X1 and Arabidopsis SGS3, XS domain, is around 140 amino acid residues in length. The XS domain contains an absolutely conserved aspartate that could present an enzymatic active site (Bateman 2002). The XS domain containing proteins are predicted by ncoils to contain coiled-coils (Lupas et al. 1991), which indicate that they could oligomerise. Like most coiled-coil proteins (which could form a dimeric or a trimeric structure), different members of the XS domain family may oligomerise via their coiled-coils to form a variety of complexes (Bateman 2002). The XH domain is between 124 and 145 residues in length. The XH domain contains one absolutely conserved glutamate which could potentially be an active site or other functionally vital region. XS and XH domains are found in most of these proteins and two domains may interact (Bateman 2002; Xie et al. 2012a). The zf-XS domain is an N-terminal cysteine/histidine zinc binding domain and usually accompanies a XS domain (Bateman 2002).

Up to now, some experimental data have been reported indicating the functions of this family, such as SGS3 in Arabidopsis, OXHS in rice and SISGS3 in tomato (Mourrain et al. 2000; Glick et al. 2008; Qin et al. 2009). SGS3 is essential for post-transcriptional gene silencing and natural virus resistance (Mourrain et al. 2000; Xie et al. 2012b), which are vital for endogenous gene silencing from DNA viruses (Muangsan et al. 2004; Qin et al. 2009) and the production of trans-acting siRNAs in Arabidopsis (Peragine et al. 2004). SGS3 acts upstream of RDR6-dependent dsRNA production (Yoshikawa et al. 2005) and the XS domain probably acts as an RNA recognition motif (Bateman 2002; Zhang and Trudeau 2008; Elmayan et al. 2009). Along with the function of SGS3 in Arabidopsis antiviral response, SISGS3 is targeted by the tomato yellow leaf curl geminivirus PTGS suppressor protein V2 (Glick et al. 2008). Nine rice OXHS genes are responsive to at least one of the abiotic stresses including salt, drought, cold, and abscisic acid treatment (Qin et al. 2009).

These reports in plants suggested that XHS family genes may be crucial not only for plant development but also for response and adaptation to stresses. So, in order to indicate the functions of ZmXHS gens in this family, transcript expression levels of the family genes were investigated in various maize tissues and in the seedling leaves under various abiotic and biotic stresses. The results presented in this study gave an important reference for function studies of XHS family genes in maize.

Materials and methods

Isolation of XHS gene in maize

The sequences of 14 Arabidopsis and 11 rice XHS proteins were downloaded from the TAIR database (http://www.arabidopsis.org) and TIGR database (http://rice.plantbiology.msu.edu) (Qin et al. 2009). To obtain all the XHS genes in maize, BLASTP searches were performed in the Maize sequence database (http://www.maizesequence.org/index.html) and Gramene database (http://www.gramene.org/Multi/blastview) with the Arabidopsis and rice XHS proteins as queries. First, all the corresponding protein sequences of the putative XHS family members were downloaded if it satisfied with E < 10-10 and including XH domain (accession no. PF03469), XS domain (accession no. PF03468), or zf-XS domain (accession no. PF03470). And then, all the candidate proteins were confirmed with the Pfam database (http://www.sanger.ac.uk/Software/Pfam/search.shtml) (Sonnhammer et al. 1997). The full-length cDNA sequences of ZmXHS genes in maize were downloaded from the Maize sequencing database.

Gene structure analysis of maize XHS genes

The information of all ZmXHS genes, including chromosomal location, full-length DNA sequences were obtained from the B73 maize sequencing database (http://www.maizesequence.org/index.html). Exons and introns structures of ZmXHS genes were confirmed by using GSDS (http://gsds.cbi.pku.edu.cn/) (Guo et al. 2007).

Motif display analysis of ZmXHS proteins

Multiple Expectation Maximization for Motif Elicitation (MEME) utility program (Bailey et al. 2009) was used to display domains of ZmXHS, OsXHS, AtXHS, and SbXHS proteins. The matrix for phylogenetic analysis included the 14, 11, 19, and 10 XHS genes from Arabidopsis, rice, sorghum and maize.

Promoter regions analysis of ZmXHS genes

To investigate cis-elements in promoter sequences of ZmXHS genes, 2 kb of B73 genomic DNA sequences upstream of the initiation codon (ATG) were downloaded from NCBI. Promoter structure was predicted using Promoter 2.0 software (http://www.cbs.dtu.dk/services/Promoter/) and PLACE (Plant cis-acting Regulatory DNA Elements, with more than 6 bp) (http://www.dna.affrc.go.jp/PLACE/) (Higo et al. 1999).

Yeast two-hybrid assays

A Gal4-based two-hybrid system was used as described by the manufacturer (Clontech). The full-length and truncated ZmXHS5 (ZmXHS5-1, lacking the XH domain; ZmXHS5-2, XH domain alone) coding region were amplified by PCR with primers containing restriction sites and cloned into the pGADT7 cloning vector (Clontech) to make the bait plasmid pGADT7-ZmXHS. The coding sequences of the ZmXHS2 and ZmXHS5 were cloned into the DNA binding domain vector pGBKT7. The primers and restriction sites used to create these constructs are listed in Supplementary Table 1. Each pGBKT7-ZmXHS was separately co-transformed with pGADT7-ZmXHS5/ZmXHS5-1/ZmXHS5-2 into Saccharomyces cervisiae AH109 by the LiAc-mediated yeast transformation method according to the Yeast Protocols Handbook of Clontech. Co-transformed yeast cells were selected on synthetic medium lacking Leu and Trp and interaction was selected on synthetic medium lacking Leu, Trp, His, and Ade.

Plant materials stress treatment

Seeds of the maize (Zea mays) B73, were surface-sterilized, germinated and hydroponically grown to the three-leaf stage in pots filled with vermiculite, with four seeds per pot. The pots were placed in a greenhouse at 28 °C under 16 h light and 20 °C under 8 h dark and watered once every 3 days. For ABA treatment, leaves were sprayed with 100 μM (+)-cis, trans-ABA and then rapped up with preservative film, followed by sampling at 0, 1, 3, 6, 12, and 24 h. Abiotic treatments concluded cold, NaCl and PEG. For cold stress, the seedlings were put into a growth chamber at 4 °C and sampled at 0, 1, 3, 6, 12, and 24 h, respectively. For NaCl and PEG treatment, the seedlings were carefully removed from the vermiculite and washed clean with tap water and then dried using filter paper. Roots of the seedlings were then submerged in 200 mM NaCl or 20 % PEG (molecular weight 6,000) solution, air conditioned by a pump and sampled at 0, 1, 3, 6, 12, and 24 h, respectively (Zhang et al. 2012).

Microarray data collection and analyses of expression profiles

The expression behaviors of ZmXHS genes were examined in a set of maize transcriptome data at PLEXdb (http://www.plexdb.org). The microarray data of genome-wide gene expression datas of maize inbred line B73 (GSE27004) and the data of transcriptomic analysis of induced senescence in maize were provided by Kaeppler from University of Wisconsin (Sekhon et al. 2011, 2012). Gene expression data from the Affymetrix GeneChip array data during three kinds of fungal infection were downloaded from GEO with accession numbers, GSE31188, GSE19501, and GSE29747, respectively (Ghareeb et al. 2011; Voll et al. 2011). The data were analyzed by GeneSpring 12.5 software. Heat map was used to present the number of ZmXHS genes, and the map was generated using MultiExperiment Viewer (MeV, version 4.8.1) software. The data were adjusted by median centering of genes. The data were clustered by complete linkage clustering method, and a euclidean distance metric was used.

Transcript level analysis ZmXHS genes

Total RNA was prepared using the TRIZOL reagent (Invitrogen, Karlsruhe, Germany) and then purified using the DNase I (RNase free) (TaKaRa, Dalian) according to the manufacturer's protocol. After an initial photometric determination of total RNA concentration, ethidium bromide fluorescence of ribosomal bands on the electrophoresis-gel was used for the control of total RNA integrity and the adjustment of equal RNA amounts. Real-time PCR was performed in an optical 96-well plate with an ABI StepOnePlus Real-Time PCR System. Gene-specific primers were designed for all ten ZmXHS genes (Table 2) and ZmGAPDH transcript served as the control (Kozak 1999). Each reaction contained 10 μl of SYBR Premix Ex Taq (TaKaRa, Dalian), 1.0 μl of cDNA samples, 0.4 μl ROX Reference Dye II and 10 μM gene-specific primers in a final volume of 20 μl. The thermal cycle used was as follows: 95 °C of 30 S, 40 cycles of 95 °C for 5 s and 60 °C for 60 s. For melt curve, 95 °C 15 S, 60 °C for 60 s and 95 °C 15 S. The analysis of real-time PCR data used 2−ΔΔC T method (Livak and Schmittgen 2001).

Table 1 List of X1-homologous genes in maize
Full size table
Table 2 Primers of maize X1-homologous genes for real-time PCR
Full size table

Results

Identification of XHS family genes in maize

After carefully surveying the maize genome, ten members were defined as ZmXHS genes (Table 1). There was no ZmXHS gene homologous to OXHS3 and OXHS10 but two OXHS8 homologous genes named ZmXHS8a and ZmXHS8b. The length of ZmXHS proteins ranged from 253 aa (amino acids) to 691 aa. BLAST analysis against the Pfam database showed that all of them including XH domain (accession no. PF03469), XS domain (accession no. PF03468), or zf-XS domain (accession no. PF03470) (Table 1).

The ten ZmXHS genes were distributed on seven maize chromosomes: chromosome 1 contained two genes; chromosome 3 contained three genes, and chromosome 4, 6, 8, 9, 10 contained one gene, respectively (Fig. 1). Based on phylogenetic results (Supplementary Fig. 1), three paralogs were identified in ZmXHS genes including ZmXHS1/ ZmXHS2, ZmXHS6/ZmXHS7, and ZmXHS8a/ZmXHS8b (Fig. 1). The full-length cDNA sequences were compared with the corresponding genomic DNA sequences to determine the numbers and positions of exons and introns within each ZmXHS gene by using GSDS (http://gsds.cbi.pku.edu.cn/chinese.php) (Guo et al. 2007). The genome structure of the ZmXHS genes showed that most ZmXHSs had six exons, except for ZmXHS8a, 8b, 9 which had three (Fig. 2; Table 2).

Fig. 1
figure 1

Chromosomal localization of maize XHS genes. Segmental duplicates, including ZmXHS1/ ZmXHS2, ZmXHS6/ZmXHS7, and ZmXHS8a/ZmXHS8b

Full size image
Fig. 2
figure 2

Gene structure of maize XHS gene family. Exons and introns were showed by filled boxes and single lines, respectively

Full size image

Motif analysis and protein architecture

To identify the conserved domains distribution in XHS proteins, the MEME web server was employed to analyze the protein sequences from Arabidopsis, rice, sorghum and maize. Three putative conserved domains were detected in the ZmXHS family, including XH (PF03469), XS (PF03468), and zf-XS (PF03470) which have been identified in the X1 and SGS3 proteins. According to the configuration of the putative domains, the 10 ZmXHS proteins can be divided into four types (Fig. 3). The protein sequences in type I, including ZmXHS1, ZmXHS2, ZmXHS4, ZmXHS5 and ZmXHS6 contained the XH domain, XS domain, and zf-XS domain. Three proteins ZmXHS8a, ZmXHS8b, ZmXHS9 each containing only the zf-XS domain were classified as type II. Type III protein ZmXHS7 contained XH domain and XS domain, while Type IV protein ZmXHS11 contained XS domain and zf-XS domain.

Fig. 3
figure 3

Putative motif distribution in maize XHS proteins. Domains of XHS proteins were investigated by MEME web server. Motif1 XH; Motif2 XS; Motif3 zf-XS

Full size image

All these three putative conserved domains were present in most of the XHS members of rice, Arabidopsis and sorghum. According to the configuration of the putative domains, the XHS proteins of rice, Arabidopsis and sorghum can be divided into three or four types, respectively (Supplementary Fig. 2) (Qin et al. 2009).

The XH domain of ZmXHS5 is necessary for interacts with itself and ZmXHS2

Our previous study indicated that ZmXHS5 interacts with itself and ZmXHS2 (Supplementary Fig. 3). To identify the protein domains of ZmXHS5 responsible for the interaction, we generated two truncation mutants of ZmXHS5 in pGADT7 (Fig. 4a): lacking the XH domain (ZmXHS5-1), the XH domain alone (ZmXHS5-2). We checked the interaction of these truncated ZmXHS5 mutants with full-length ZmXHS5 and ZmXHS2 using the yeast two-hybrid assay described above. ZmXHS5-2 was able to interact with ZmXHS5 and ZmXHS2, because co-transformation of the two pairs enabled yeast cell to grow in the absence of Ade and His (Fig. 4b). However, ZmXHS5-1 did not interact with ZmXHS5 and ZmXHS2. These results indicated that the XH domain of ZmXHS5 is necessary for ZmXHS5–ZmXHS5 and ZmXHS5–ZmXHS2 interactions.

Fig. 4
figure 4

The XH domain mediates ZmXHS5–ZmXHS5 and ZmXHS5–ZmXHS2 interactions a Schematic structure of the full-length and truncated ZmXHS5 proteins used for yeast two-hybrid assays. ZmXHS5-1, truncated ZmXHS5 protein lacking the XH domain; ZmXHS5-2, XH domain alone; b interaction analyses of truncated ZmXHS5 proteins with ZmXHS5 and ZmXHS2 in yeast AH109 cells. The paired AD and BD fusion constructs were co-transformed into yeast. Positive clones selected on –Leu/–Trp were spotted on –Ade/–Leu/–Trp/-Ade medium

Full size image

cis-Element analysis

By searching the PLACE database, promoter regions (2kp range B73 genomic DNA sequences upstream of translation start site) of ZmXHS genes were analyzed. Amounts up to 58 putative cis-elements more than 6 bp length were identified (Supplementary Table 2). In this study, a series of cis-elements were found that involved in abiotic stress responsive, phytohormone, pollen-specific and quantitative, seed development and germination, light and circadian rhythms regulation, Ca2+-responsive, root hair cell-specific, and CO2-responsive transcriptional activation.

Among these cis-elements, five kinds were distributed in all the ten ZmXHS genes, i.e., the dehydration-stress responsive element MYBCORE and MYCCONSENSUSAT (Solano et al. 1995; Abe et al. 2003), Ca2+-responsive element CGCGBOXAT (Yang and Poovaiah 2002) and EECCRCAH1 (Guo et al. 2010), ABA-responsive element DPBFCOREDCDC3 (Kim et al. 1997; Finkelstein and Lynch 2000; Lopez-Molina and Chua 2000), light-responsive element EBOXBNNAPA and INRNTPSADB (Stalberg et al. 1996; Hartmann et al. 2005), SA-responsive element GT1CONSENSUS (Villain et al. 1996; Le Gourrierec et al. 1999). Another widely distributed cis-element ABRERATCAL (Kaplan et al. 2006), CO2-responsive element was present in nine of the ten ZmXHS genes. Pollen-specific and quantitative element QELEMENTZMZM13 (Hamilton et al. 1998), phytochrome regulation element REBETALGLHCB21 (Degenhardt and Tobin 1996), root hair cell-specific element RHERPATEXPA7 (Kim et al. 2006), early responsive to dehydration element MYCATERD1 (Tran et al. 2004), fermentative pathway responsive element ANAERO1CONSENSUS (Bratić et al. 2009), also exist in most of the ZmXHS gene promoters.

By controlling efficiency of the promoters, cis-elements played essential function in the regulation of gene expression. Studies on cis-elements could provide vital foundation for further functional research of the ZmXHS gene family.

Expression profiles of ZmXHS family in different tissues and organs

In order to identify the spatial and temporal specific expression patterns of ZmXHS genes, we explored microarray data which record the gene expression levels of 60 tissues varying developmental stages of the maize (Sekhon et al. 2011). It can be seen from the heat map that all of the ten detected genes were involved in numerous biological processes, and expressed in almost tissues, but their expression levels were distinct (Fig. 5). ZmXHS2 had higher expression in reproductive organs such as cob, tassel, whole seed (DAP), endosperm (DAP), and embryo (DAP). The transcript level of ZmXHS4 was detected at the higher level in silks, anthers, sheath, outer husk, and different leaves. ZmXHS8a and ZmXHS9 had similar expression pattern but different from that of ZmXHS8b. ZmXHS8b was involved in the development of vegetative organs such as primary root, stem, SAM, sheath, internode, and innermost husk, while ZmXHS8a and ZmXHS9 had higher expression both in reproductive and vegetative organs, i.e., seed, endosperm, embryo, leaf, stem, and husk. ZmXHS5-7 and ZmXHS11 had similar expression pattern, appearing to be lowly expressed among vegetative organs and moderate expressed among reproductive organs (Fig. 5).

Fig. 5
figure 5

Organ-specific expression patterns of ZmXHS genes detected in the microarray data. Log2 ratios of expression were used to make this heat map. Red color indicates higher expression while green color signifies lower expression in 60 different tissues

Full size image

Expression profiles of ZmXHS genes under abiotic stresses

Many plant gene families were involved both in stress and development responses. In order to check whether ZmXHS genes were responsive to stresses in seedling stage, quantitative real-time PCR (qRT-PCR) was conducted to analyze all ten genes' transcriptional expression in shoots at the three-leaf stage treated by exogenous ABA, PEG, NaCl, and low temperature (4 °C).

The results indicated that, except for ZmXHS9, the other nine genes were induced in the seedling leaves by at least one of the four stresses applied (Fig. 6). Among them, six genes (ZmXHS1, ZmXHS2, ZmXHS4, ZmXHS7, and ZmXHS11) were obviously induced by PEG stress with distinct patterns. For example, the transcript levels of ZmXHS1, ZmXHS7, and ZmXHS11 were increased at the early stage of PEG stress and then decreased, while the expression level of ZmXHS2 and ZmXHS4 increased gradually and reached the highest level at the late stage of the stress. ZmXHS2, ZmXHS5, and ZmXHS6 were induced by salt stress, while ZmXHS1 and ZmXHS8b were suppressed by salt stress at 1 h after treatment. Under ABA stress condition, the expression level of ZmXHS4 increased gradually and peaked at 12 h, while that of the other nine gsenes were no obviously initiated. There was only one gene, ZmXHS8b responsive to low temperature (4 °C) treatment. The expression of ZmXHS8b was evidently induced within 1 h after low temperature treatment.

Fig. 6
figure 6

Expression levels of maize XHS genes under each treatment, including ABA, cold, NaCl and PEG stress, based on real-time quantitative PCR. The X-axes are treatment time points and the Y-axes are scales of relative expression level. The transcript level at time 0 h (untreated) was used as the calibrator and was given as 1

Full size image

Expression profiles of ZmXHS genes under biotic stresses

To discover the ZmXHS (only seven genes were detected) genes involved in biotic stresses, we detected differentially expressed genes with microarray data. Data sets of three experiments under the pathogen treatments of Fusarium moniliforme, Sphacelotheca reiliana, and Colletotrichum graminicola infection have been analyzed. As shown in Fig. 7, ZmXHS2 and ZmXHS11 were downregulated after the infection of the three pathogens. ZmXHS5 and ZmXHS8b were suppressed by C. graminicola and S. reiliana, respectively. ZmXHS7 was suppressed by F. moniliforme and C. graminicola while induced by S. reiliana treatment. ZmXHS1 was upregulated while ZmXHS9 was downregulated by C. graminicola (Fig. 7).

Fig. 7
figure 7

Expression levels of maize XHS genes under biotic stresses. CK1: Samples from uninfected control plants were taken at the same time points of T1. T1 Samples from infected leaves were taken at 36 h post infection. CK2 Samples from uninfected control plants were taken at the same time points of T2. T2 Samples from infected leaves were taken at 96 h post infection

Full size image

Discussion

X1-homologous genes (XHS) encode plant specific proteins containing three basic domains (XH, XS, zf-XS) (Qin et al. 2009). In this study, ten genes belonging to the XHS in maize were identified. Among the better analyzed plant XHS families, Arabidopsis has likely 14 XHS, rice has at least 11 expressed XHS (Qin et al. 2009), sorghum at least 19 XHS (http://www.gramene.org/Multi/blastview). The genome structure of the ZmXHS genes showed that most them had six exons, except for ZmXHS8a, 8b, 9 which had three (Fig. 2). Most rice and sorghum XHS genes also had six exons, but most Arabidopsis XHS genes had seven or eight exons (Supplementary Fig. 4). Five of ten proteins, ZmXHS1, ZmXHS2, ZmXHS4, ZmXHS5 and ZmXHS6 contained the XH domain which was necessary for proteins interaction. In Arabidopsis, the XH domain of FDM1 was necessary for FDM1–FDM1 and FDM1–IDN2 interactions (Xie et al. 2012a). In our study, the XH domain of ZmXHS5 was necessary for interacts with both itself and ZmXHS2 (Fig. 4).

Increasing evidence indicated that some gene families had vital roles both in development and response to stress (Qin et al. 2009). Examples were the genes from CBL-interacting protein kinase (CIPK) and F-BOX family and many transcription factor families such as NAC, MYB, and bZIP (Urao et al. 1993; Shi et al. 1999; Liu et al. 2000; Durfee et al. 2003; Hu et al. 2006; Kim 2006).

The members of XHS gene family in rice showed tissue-specific expression patterns. All the 11 OXHS genes were observably expressed in floral organs, and some were expressed in a wide range of different organs in rice (Qin et al. 2009). Like that in rice, the transcript levels of the ten ZmXHS genes were all had different expression in reproductive and vegetative organs (Fig. 5). The XS domain containing proteins are involved in a wide range of processes such as viral defense and stress response (Qin et al. 2009). In Arabidopsis, SGS3 was required for post-transcriptional gene silencing, natural virus resistance and sgs3 mutant shown enhanced susceptibility to CMV (Mourrain et al. 2000). In tomato, SISGS3 was specifically required for the RNA-silencing defense against geminiviruses (Glick et al. 2008). In our work, most of ZmXHS were up or down regulated by the pathogen treatments of F. moniliforme, S. reiliana, and C. graminicola. In rice, nine OXHS genes were responsive to at least one of the abiotic stresses including drought, salt, cold, and abscisic acid treatment. Over-expression of the gene OXHS2 in rice resulted in reduced tolerance to salt and drought stresses (Qin et al. 2009). In our study, except ZmXHS9, the other nine genes were induced in the seedling leaves by at least one of the four stresses PEG cold, NaCl, or ABA (Fig. 6). In cluster ІII of phylogenetic tree (Supplementary Fig. 5), ZmXHS2 was the closest homologue to OXHS2, and the both proteins have strong similarity in domain composition, suggested that ZmXHS2 might be an orthologue of OXHS2 in maize. Both genes were induced by drought and salt treatment (Fig. 6) (Qin et al. 2009). These results indicated that ZmXHS2 might have the similar function to OXHS2.