Identification and characterization of the MADS-box genes highly expressed in the laticifer cells of Hevea brasiliensis

MADS-box transcription factors possess many functions in plant reproduction and development. However, few MADS-box genes related to secondary metabolites regulation have been identified. In Hevea brasiliensis, natural rubber is a representative cis-polyisoprenoids in secondary metabolism which occurs in the rubber laticifer cells, the molecular regulation basis of natural rubber biosynthesis is not clear. Here, a total of 24 MADS-box genes including 4 type I MADS-box genes and 20 type II MADS-box genes were identified in the transcriptome of rubber tree latex. The phylogenetic analysis was performed to clarify the evolutionary relationships of all the 24 rubber tree MADS-box proteins with MADS-box transcription factors from Arabidopsis thaliana and Oryza sativa. Four type I MADS-box genes were subdivided into Mα (3 genes) and Mβ (1 gene). Twenty type II MADS-box genes were subclassified into MIKC* (8 genes) and MIKCc (12 genes). Eight MADS-box genes (HblMADS3, 5, 6, 7, 9, 13, 23, 24) were predominant expression in laticifers. ABA up-regulated the expression of HblMADS9, and the expression of HblMADS3, HblMADS5, HblMADS24 were up-regulated by MeJA. The function of HblMADS24 was elucidated. HblMADS24 bound HbFPS1 promoter in yeast and HblMADS24 activated HbFPS1 promoter in tobacco plants. Moreover, we proposed that HblMADS24 is a transcription activator of HbFPS1 which taking part in natural rubber biosynthesis.

www.nature.com/scientificreports www.nature.com/scientificreports/ occurs on the surface of the rubber particle in the rubber laticifer cells 38,39 . NR is biosynthesized by sequential condensations of isopentenyl diphosphates which are synthesized via the mevalonate pathway [40][41][42] . In the rubber tree, farnesyl diphosphate synthase (FPS) is an important enzyme in isoprenoids secondary metabolism. HbFPS1 expressed obviously in the laticifers which is possible to involve in NR biosynthesis. However, HbFPS2 and HbFPS3 have no cell-type specific expression, and they are likely to act as housekeeping nature to involve in isoprenoid biosynthesis 43 .
In H. brasiliensis, the general NR biosynthesis metabolic pathway is now clear, but the molecular regulation of some NR biosynthesis-related genes is limited 41,44 . To date, few MADS-box genes related to NR biosynthesis have been identified. For instance, three MADS-box genes of H. brasiliensis were identified. They were differentially expressed in the laticifer cells 45 . HbMADS4 was identified to down-regulate the expression HbSRPP involved in NR biosynthesis 46 . In the present study, we identified and systematically analyzed the 24 MADS-box family genes (named HblMADS1 to HblMADS24) from the rubber tree latex transcriptome. Eight MADS-box genes were identified as predominantly expressed in laticifers. Furthermore, HblMADS24 positively regulated the HbFPS1 expression.

Results
Identification and phylogenetic analysis of the MADS-box genes from H. brasiliensis. Our laboratory had obtained the H. brasiliensis latex transcriptome database by the Illumina HiSeq 2000 method 47 . A total of 36 MADS-box unigenes were obtained by scanning the H. brasiliensis latex transcriptome database. These MADS-box unigenes were used as queries in BLAST against the local H. brasiliensis genome database. A total of 24 MADS-box genes were confirmed from H. brasiliensis genome. These MADS-box genes were designated as HblMADS1 to HblMADS24, respectively (Additional file Table S1). The number of nucleotides of all the 24 MADS-box genes coding domain sequence varied from 522 bp to 1101 bp, the number of amino acids encoded the 24 MADS-box proteins varied from 173 aa to 366 aa, and the predicted relative molecular mass ranged from 20.4 to 41.18 kDa, with protein isoelectric point (pI) in the range of 5.04 to 10.14 (Additional file Table S1).
To determine the evolutionary relationships between these MADS-box genes in rubber tree latex and other species, the phylogenetic tree was constructed among MADS-box genes from H. brasiliensis and known MADS-box proteins from Arabidopsis thaliana and Oryza sativa using the neighbor-joining method (Fig. 1). According to the phylogenetic analysis, 24 MADS-box genes from H. brasiliensis were classified into two groups, including type I MADS-box genes (4 genes) and type II MADS-box genes (20 genes). Four type I MADS-box genes were subdivided into Mα (3 genes) and Mβ (1 gene). Twenty type II MADS-box genes were subdivided into MIKC* (8 genes) and MIKC c (12 genes). The 12 MIKC c genes were further classified into 5 subfamilies: SOC1 (4 genes), AGL17 (3 genes), SVP (2 genes), AP1 (2 genes) and FLC (1 gene).

Gene structure analysis and identification of conserved motifs of H. brasiliensis MADS-box genes.
The exon/intron patterns of MADS-box genes from H. brasiliensis were analyzed by the online software GSDS. The genes of the same subfamily had significant similar exon-intron structures, differing only in the number of nucleotides of exon/intron (Fig. 2). In general, the type II MADS-box genes had more exons compared with the type I MADS-box genes, which suggested that the type II MADS-box genes have more complex gene structure. The number of exon of the twenty type II MADS-box genes ranged from 7 to 11, while four type I MADS-box genes contained only one exon. The MIKC* genes displayed less number of nucleotides and more exons than the MIKC C genes.
The conserved motifs of 24 HblMADS proteins were analyzed to illuminate the features of MADS-box protein sequences by MEME motif search tool, resulting in the identification of 17 conserved motifs (Fig. 3). In the same subfamily, most of the closely related proteins distributed similar motif type. All the 24 HblMADS proteins contained motif 1 which is the most typical MADS-box domain in plant MADS-box proteins. Motif 3 represented the most conserved K domain, which was verified in all the type II HblMADS proteins. The K-domain was also only observed in other plants type II MADS-box proteins 48 .

Expression analysis of MADS-box genes in the latex in response to hormone treatments. Eight
MADS-box genes (HblMADS3, 5,6,7,9,13,23,24) maintained higher expression levels in latex. However, HblMADS6, HblMADS7, HblMADS13, HblMADS23 have been identified 45,46 . HblMADS3, HblMADS5, HblMADS9, HblMADS24 were analyzed in response to exogenous plant hormones. The expression patterns of these four genes were different under abscisic acid (ABA), ethrel (ET), methyl jasmonate (MeJA), and salicylic acid (SA) treatments (Fig. 5). The results indicated that MeJA treatment up-regulated the expression of HblMADS3, HblMADS5 and HblMADS24 at either 9 h or 12 h time points, while had no influence on that of HblMADS9. ABA treatment evidently up-regulated the expression of HblMADS9 at 12 h, whereas had not significantly affected that of HblMADS3, HblMADS5 and HblMADS24. Under the SA treatment, the expression of HblMADS9 and HblMADS24 were slightly up-regulated at 9 h or 6 h time point, while had no significantly effect on that of HblMADS3 and HblMADS5. ET stress had no obvious influence on the expression of four HblMADS genes. www.nature.com/scientificreports www.nature.com/scientificreports/ Subcellular localization of HblMADS24. To elucidate the function of HblMADS24, we performed the subcellular localization analysis on HblMADS24. We found that the green fluorescent protein (GFP) tagged HblMADS24 was indeed present in the nucleus of onion epidermal cells, while GFP alone exhibited a cytoplasmic distribution (Fig. 6).
Activation of the HbFPS1 promoter by HblMADS24 in yeast. HbFPS1 expressed obviously in the laticifers which is possible to involve in natural rubber biosynthesis 43 . The yeast one-hybrid analysis was performed to investigate whether HblMADS24 binds the HbFPS1 promoter. The yeast clones harboring pGAD-HblMADS24 and pHis-pHbFPS1 could grow on triple dropout medium lacking histidine, tryptophan, and leucine (SD/-Trp/-His/-Leu) containing 70 mM 3-amino-1, 24-triazole (3-AT), suggesting HblMADS24 bound the HbFPS1 promoter in yeast (Fig. 7).
Activation of the HbFPS1 promoter by HblMADS24 in plants. Since HblMADS24 was able to interact with the HbFPS1 promoter in yeast, the Dual-LUC method was used to investigate whether HblMADS24 can regulate the HbFPS1 promoter in plants. The reporter strain pGreen-pHbFPS1 and effector strain pGreenII62Sk-HblMADS24 were mixed and injected into tobacco leaves for Dual-LUC assays. The HbFPS1 promoter drove luciferase expression weakly alone, while the HblMADS24 expression induced an obvious increase in luciferase activity (Fig. 8). The result indicated that HblMADS24 activated the HbFPS1 promoter expression. www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
The MADS-box genes have been identified and characterized in some plants species, such as Arabidopsis thaliana 3 , Raphanus sativus 9 , Oryza sativa 49 , Populus trichocarpa 50 , Zea mays 51 , Brassica rapa 52 , Vitis vinifera 53 , Malus domestica 54 , Prunus mume 55 . H. brasiliensis is a well-known rubber-producing plant used to produce commercially valuable natural rubber 56 . However, the characterization and systematically analysis of the MADS-box genes family has not been performed in H. brasiliensis.
In this study, we comprehensive analyzed 24 MADS-box family genes from the rubber tree latex transcriptome, including phylogenetic analysis, gene structures, conserved motifs distribution, expression patterns in different tissues and in response to hormone treatments, gene functional analysis. 24 MADS-box genes were classified into 8 subgroups, which provided a firm basis for better realizing the function of MADS-box genes. In addition, the function of the protein will probably change because of the different exon/intron patterns 57 . Twenty type II MADS-box genes had 7 to 11 exons, while four type I MADS-box genes had only one exon, which could suggest that the type II MADS-box genes contained more variable and complex function. The similar results had also been observed in Arabidopsis thaliana 3 , Chinese cabbage 52 , apple 54 , Prunus mume 55 , soybean 58 .  www.nature.com/scientificreports www.nature.com/scientificreports/ The expression patterns of all the 24 MADS-box genes were detected in five different tissues (roots, barks, leaves, flowers, latex) by qRT-PCR. A heat map showed that most MIKC* genes were highly expressed in flowers, which were consistent with previous studies in Arabidopsis thaliana and Oryza sativa 14,59 . However, most MIKC C genes were highly expressed in leaf and latex. Type I displayed similar or distinct expression profiles. For instance, HblMADS24 was more expressed in latex, whereas HblMADS22 was uniquely expressed in flower, HblMADS10 and HblMADS21 was more expressed in leaf, which demonstrated that these genes may have multiple functions.
Phytohormone plays key role in NR biosynthesis 60 . Laticifers differentiation is specifically induced by JA 61 . Moreover, JA up-regulated NR biosynthesis-related genes expression, suggesting NR biosynthesis in laticifers is regulated by JA signalling 60,[62][63][64] . ET has been widely applied to stimulate rubber production 65,66 . ABA treated rubber trees exhibited to increases in latex yield 67 . ABA markedly up-regulated NR biosynthesis-related genes expression, suggesting NR biosynthesis in laticifers is also regulated by ABA signalling 68 . SA could also induce a transient increase latex yield 67 . How these hormones are connected to the NR biosynthesis pathway and how their action is integrated with other regulatory circuits is currently unknown. In our present study, ABA up-regulated the expression of HblMADS9. NR biosynthesis was probably regulated by JA signaling in laticifers 69,70 . MeJA regulated the expression of HblMADS3, HblMADS5, HblMADS24, which indicated HblMADS3, HblMADS5, HblMADS24 may play an important role in JA signaling pathway. But the regulatory mechanism of NR biosynthesis is not clear 71,72 . It will be of great interest to elucidate whether MADS-box transcription factors can regulate NR biosynthesis with JA and ABA as the regulatory signals.
Compared to type II MADS-box genes, the data about type I MADS-box genes is inadequate 73 . Previous studies indicated that Arabidopsis type I MADS-box genes are involved in plant development and reproduction [73][74][75][76] . Little information is available on type I MADS-box participating in the regulation of secondary metabolism. In our present study, HblMADS24 is a typical I MADS-box gene that bound the HbFPS1 promoter. Moreover, HblMADS24 activated the HbFPS1 promoter, suggesting HblMADS24 maybe participate in the regulation of natural rubber biosynthesis. As a result, over-expression of HbFPS1 driven by HBMADS24 would increase latex yield. Identification of the MADS-box genes highly expressed in H. brasiliensis laticifers cells would greatly help to uncover the molecular regulation basis of natural rubber biosynthesis.     61 . For each hormonal treatment, the latex samples were collected and mixed from one group of ten-trees when treated at 1, 3, 6, 9, 12, 24 and 48 h 77 . One group of ten-trees without any treatment was as control. All the samples were quickly stored in the RNA extraction buffer. Four other tissues of rubber tree: roots, barks, leaves, and flowers were sampled for RNA extraction.

Identification of the MADS-box genes in the laticifer cells.
The local H. brasiliensis genome database had been established using the rubber tree genome data 72,78 and NCBI-Blast-2.2.28+-win32 software in our previously study 79 . A total of 36 MADS-box unigenes were obtained in the rubber tree latex transcriptome database 47 . The MADS-box unigenes were used as queries in searching for MADS-box genes loci in the local genome of H. brasiliensis using the BLASTx. Finally, MADS-box genes were obtained in the local H. brasiliensis genome database according to the method previously 80 . The molecular weight and pI of each HblMADS protein were predicted using ExPASy database (http://web.expasy.org/compute_pi/) 81 .
Phylogenetic analysis. The MADS-box transcription factor protein sequences from Arabidopsis and O. sativa were downloaded from the phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html) 82 . The phylogenetic tree was constructed among MADS-box proteins from H. brasiliensis and known MADS proteins from Arabidopsis and O. sativa according to the method previously 80 .
Gene structure analysis and identification of conserved motif. The online software GSDS (http:// gsds.cbi.pku.edu.cn/) 79 was utilized to reveal the exon-intron structure and coding domain sequences (CDS) of MADS-box genes from H. brasiliensis. The MEME (http://meme-suite.org/tools/meme) 80 was employed to analyze the conserved domains of HblMADS proteins.

Expression analysis of the MADS-box genes.
Latex total RNA was extracted as described previously 77 , and total RNAs from roots, barks, leaves, and flowers were isolated according to Li's method 46 . The first-strand cDNA was synthesized in accordance with the manual of the RevertAid TM First-Strand cDNA Synthesis Kit (Fermentas, Lithuania). The quantitative real-time PCR (qRT-PCR) was performed in accordance with the manual of the SYBR Premix EX Taq Kit (TaKaRa, Japan). The HbACT7 was amplified as the standard control 24 . The primers of MADS-box genes from H. brasiliensis for RT-qPCR were listed in Additional file Table S2. The qRT-PCR reaction conditions were as follows: pre-heating at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s, 58 °C for 20 s, and 72 °C for 25 s. Three individual biological reactions were replicated. The relative expression levels were analyzed using the Stratagene Mx3005P Real Time Thermal Cycler software (Agilent, America) 79 .
Subcellular localization of HblMADS24. The CDS of HblMADS24 was fused in the pCAMBIA1302 vector to generate pHblMADS24-GFP. The amplified primers were listed in Additional file Table S2. The pHblMADS24-GFP or pCAMBIA1302 was individual introduced into onion epidermal cells by Agrobacterium-mediated method. The transformed onion epiderm was placed on Murashige Skoog solid medium in darkness at 26 °C. After culturing for 5 h, onion epiderm was observed under a confocal microscope (Zeiss LSM510, Germany).
Transcriptional activation. The HbFPS1 promoter with 975 bp nucleotides that was cloned into pHiS2.1 vector (Clontech), generating bait vector pHis-pHbFPS1. The amplified primers based on described previously 83 . The CDS of HblMADS24 was fused into pGAD7 vector to generate prey vector pGAD-HblMADS24. The