Genome-wide analysis of physic nut MADS-box gene family and functional characterization of the JcMADS05 gene in transgenic rice

Background: Physic nut ( Jatropha curcas ), a non-edible oilseed plant, is among the most promising alternative energy sources because of its high seed oil content, rapid growth and extensive adaptability. Proteins encoded by MADS-box genes are important transcription factors involved in the regulation of plant growth, seed development and responses to abiotic stress. However, there has been no in-depth research on the MADS-box genes and their roles in physic nut. Results: In the present study, 63 MADS-box genes ( JcMADSs ) were identified in the physic nut genome, and classed into five groups (MIKC, Mα, Mβ, Mγ, Mδ) based on phylogenetic comparison with Arabidopsis homologs. Expression profile analysis based on RNA-seq suggested that many JcMADS genes were expressed most strongly in seeds, and seven of them responded in leaves to at least one abiotic stressor (drought and/or salinity) at one or more time points. Transient expression analysis and a transactivation assay indicated that JcMADS05 is a nucleus-localized transcriptional activator. Plants overexpressing JcMADS05 did not show altered plant growth, but the overexpressing plants did exhibit reductions in grain size, grain length, grain width, 1000-seed weight and yield per plant. Further data on the reduced grain size in JcMADS05 -overexpressing plants supported the putative role of JcMADS genes in seed development. Conclusions: This study will be useful in understanding the involvement of MADS-box genes in growth and development in addition to their functions in abiotic stress resistance, and will ultimately form the basis for functional characterization and the exploitation of candidate genes for the genetic engineering of physic nut. genes in stress tolerance seed putative

lines have larger seeds compared with wild-type seeds [16]. The Arabidopsis agl62 mutant shows accelerated endosperm cellularization, showing that AGL62 is required for suppression of cellularization [17]. The svp and flm mutants exhibit temperatureinsensitive flowering across wide temperature ranges [18], while FUL has a role in leaf and fruit development and in flowering in Arabidopsis [ 19]. SEP1, SEP2, and SEP3 are critical for development of petals, stamens and carpels [20]. In addition to participating in regulating plant growth and development, evidence is accumulating to suggest that MADSbox genes are involved in plant responses to salt and drought stresses. For example, CaMADS-overexpressing Arabidopsis plants show increased resistance of cold and salt stresses [21]. Loss-of-function mutations in SVP result in sensitivity to drought stress, while the SVP-overexpressing plants are more tolerant [22]. In tomato, SlMBP11-RNAi lines are less tolerance to salinity stress, but overexpressing this MADS-box gene confers salt stress tolerance [23]. However, although various members of MADS-box family have been cloned and functionally studied, little is known about the members of this family and their roles in many taxa, including the Euphorbiaceae.
Physic nut, a small woody member of the Euphorbiaceae, is a non-food oilseed crop grown mainly in tropical and subtropical regions and its seed oil is used widely in industry [24].
Importantly, it has drawn much attention because it is suitable for biodiesel production owing to its rapid growth, ease of propagation, high oil content and extensive adaptability [24]. Further research is thus necessary in order to elucidate the molecular mechanisms that underlie the regulation of growth and development of physic nut. The recent completion of the physic nut genome sequence allows us to explore all the JcMADS genes at the genome level. However, there has as yet been no systematic study on the identification, classification, expression profiles or functions of MADS-box genes in this species. To address this deficiency, we firstly searched for and identified 63 MADS-box genes in the physic nut genome (hereafter referred to as JcMADS genes). Secondly, we investigated their phylogenetic relationships, conserved motifs, chromosomal distribution, expression profiles and potential roles in physic nut development. Finally, JcMADS05 was chosen for further functional analysis, and we tested its effects in transgenic crop. This study focuses on the functional roles of JcMADS genes in the development of physic nut.

Identification of MADS-box proteins in physic nut
All Arabidopsis MADS-box protein sequences were used as queries in a BlastP search to identify physic nut proteins. A Hidden Markov Model (HMM) search was also performed against the physic nut protein database using the MADS-domain PF03106. In total, 63 putative MADS proteins were identified in physic nut, with the presence of the MADS-box domain in each of them being confirmed by a SMART website search. These genes were named sequentially from JcMADS01 to JcMADS63 according to their chromosomal locations (Additional File 1). The 63 JcMADS genes ranged in length from 195 (JcMADS35) to 1164 (JcMADS34), thus potentially the proteins encoded would be from 64 to 387 amino acids; their GenBank accession numbers are given in Additional File 1.
As shown in Fig. 4, most of the JcMADS genes were expressed more highly in seeds at the S1 stage than at the S2 stage. It was noteworthy that nine genes (JcMADS03, 05, 12, 14, 25, 34, 46, 48 and 62) was detected expression only in seeds at S1 stage. Based on the results of expression pattern analysis, the JcMADS05 gene was chosen for functional research. P h e n o t y p i c a n a l y s i s o f t r a n s g e n i c r i c e p l a n t s e x p r e s s i n g J c M A D S 0 5 To investigate the role of JcMADS05 gene in regulating plant development, and to assess the feasibility of using JcMADS genes to control seed size in an important crop plant, we overexpressed this gene in rice. Three independent transgenic lines (OE1, OE2 and OE3) were confirmed as expressing JcMADS05 expression using semi-quantitative RT-PCR, and selected for further study. Expression of JcMADS05 were detected in transgenic lines, whereas no expression was found in WT (wild type) plants (Fig. 8C). Phenotypic analysis showed that the growth and flower structure of transgenic plants overexpressing JcMADS05 were similar to those of WT plants ( Fig. 8A and B). Statistical analysis indicated that there was no obvious difference in root and shoot lengths in the transgenic plants compared to the WT plants ( Fig. 8D and E). Taken together, these results led to the conclusion that JcMADS05 did not have any major effect on the growth of the transgenic plants.
O v e r e x p r e s s i o n o f J c M A D S 0 5 r e d u c e s t h e g r a i n s i z e i n t r a n s g e n i c r i c e As described above, JcMADS05 expression was most strongly detected in seed, suggesting that JcMADS05 might have an important role in seed growth and development. To test this, we examined the effects of JcMADS05 overexpression on rice grain size. We found that JcMADS05 transgenic plants produced dramatically smaller seeds than the WT lines ( Fig. 9A). The results also showed that JcMADS05 transgenic plants had a significant reduction in both grain length and width compared to the WT plants ( Fig. 9B and C). We also detected a significant reduction in 1000-seed weight and yield per plant in JcMADS05 transgenic lines ( Fig. 9D and E). Our data suggested that overexpressing JcMADS05 significantly altered seed size in transgenic plants.
To study the molecular mechanism of JcMADS05 gene regulates grain size, we further tested the expression of grain-size-related genes (Fig. 9F). The results showed that expression of some positive regulatory factors, such as GS2, SMG11, was significantly lower in transgenic plants than that in wild type, while expression of some negative regulatory factors, such as OsMKP1, GW2, was obviously higher than that in wild-type.
Taken together, these data supported a putative role for JcMADS genes in seed development.

Discussion
Increasing evidence suggests that the MADS-box genes are involved in a series of plant physiological phenomena. Up to now, many studies on the functions of the MADS-box genes have been focused on the model plants rice and Arabidopsis [ 27]. The molecular mechanisms involved in seed development in the biofuel plant physic nut, and more specifically the identities, expression profiles and functions of its MADS-box genes remain poorly understood. We therefore characterized and examined expression profiles of MADS-box genes in this species, and chose one (designated JcMADS05) that was most strongly expressed in seed for further functional analysis by transgenically expressing it in rice.
In the present study, a total of 63 MADS-box genes were identified in the physic nut genome. Compared with Arabidopsis (genome size 125 Mb) and rice (genome size 466 Mb), the MADS-box family seems to have relatively fewer members in the physic nut genome (genome size 320 Mb) [25]. One possible explanation for the smaller number of JcMADS genes may be that MADS-box family genes in the physic nut genome did not undergo a chromosomal segment duplication event during the early evolution of the species [25], whereas such duplications made major contributions to the expansion of both rice MADSs and Arabidopsis MADSs [ 9-10]. Our phylogenetic tree showed that there were twenty-two MADS genes in the Mβ group in Arabidopsis, whereas there were only four JcMADS genes in group Mβ (Fig. 1). These finding suggests that the members of the group may have been either acquired in the Arabidopsis lineage or lost in the physic nut after divergence from the last common ancestor shared by Arabidopsis and physic nut. Motif analysis indicated that the distribution of protein motifs across the different groups was diverse, but members of the same group had a similar motif complement (Fig. 2) [14]. Our results show that the evolution and classification of the members of MADS-box family is quite highly conserved in the physic nut, as it is in other plant species.
Preliminary predictions about the biological functions of genes and their products can be made by analysis of gene expression profiles, we therefore detected the expression of 63 MADS-box genes sequencing-based transcriptome data. Our results show that JcMADS25 expression was highest in seeds. Its Arabidopsis homolog AGL11(STK) is essential for seed development [15], and its homolog in oil palm SHELL controls seed oil yield [30]. It can therefore be inferred from the high levels of JcMADS25 expression in physic nut seeds that it may be involved in regulation of the development of physic nut seed. JcMADS16 was most highly expressed in roots, and in Arabidopsis, its homologous AGL12 is also preferentially expressed in root tissues and is essential for root development [31][32]. The results indicated that JcMADS16 may play an important in physic nut root development.
TT16, a MADS-box transcription factor, which affects seed development [33], and its homolog JcMADS05, are preferentially expressed in seeds, suggesting that JcMADS05 may have a significant regulatory role in seed development. JcMADS09 and 47 are expressed in all tissues (Fig. 4), indicating that they may participate in the overall development of the physic nut plant. It is worth noting that many JcMADS genes showed preferential expression in seeds, implying that they may all be very important for physic nut plants in seed development. Overall, we deduce that JcMADS genes may have functions in each growing stages of physic nut plants; further study is required to confirm their roles.
Research increasingly have suggested that MADS-box genes participate in responses to various abiotic stresses in many plant species [21, [27][28]. For example, CaMADS, which is strongly induced by salinity stress, and by abscisic acid, acts as a mediator that has positive feedback effects on cold and salinity stress signaling pathways in pepper [21].
OsMADS26 is a regulator of drought stress response in rice [28]. AGL22 gene plays a crucial role in connecting changes in the initiation of drought stress responses and primary metabolism [34]. Although some studies have begun to identify certain genes of the MADS-box family as important molecular components of abiotic stress responses, we have hitherto lacked integrated information about the responses of members of MADS-box family to drought and salt stresses in physic nut. In this study, RNA-based sequencing data in response to drought and salt stress, combined with qRT-PCR analysis, enabled us to identify JcMADS genes that respond to abiotic stress. For example, expression of JcMADS42, 43 and 47 was induced or inhibited by salt and drought stresses at one or more time points, whereas JcMADS22, 30 and 53 responded only to drought stress (Fig. 5).
Collectively, we preliminary judgment these JcMADS genes may play important roles in the regulation of abiotic stress responses in physic nut, and their functions merit further investigation.
Grain size, which is an important agronomic trait in many crops, is determined by grain width, grain length and grain thickness [35]. Although some genes that manipulate seed size have been identified in crop plants [36], the molecular mechanisms that regulate seed size are still poorly understood. In our work, we found that JcMADS05, a gene in group MIKC, was most highly expressed in the seeds of physic nut (Fig. 4), and in order to investigate its function we tested its effects in transgenic rice. Our results showed that the JcMADS05 transgenic plants had smaller, shorter and narrower grains and lower 1000grain weight compared with wild-type plants (Fig. 9). Furthermore, our results also suggested that JcMADS05 overexpressing plants reduced expression of GS2 and SMG11, and increased expression of OsMKP1 and GW2 (Fig. 9F). SMG11 overexpressing plants increases grain size by altering expression of several grain-size-related genes [35]. Gain of function mutations in OsMKP1 reduces grain size, conversely, results in larger grain [37]. Loss of GW2 function increase grain size and grain weight [38]. Overexpression of GS2 increases grain size and weight [39]. Collectively, JcMADS05 overexpressing plants reduces grain size, at least in part, by influencing expression of grain-size-related genes.
These findings further support a role for JcMADS05 in negative regulation of grain size.
Given that reporter gene studies in Arabidopsis protoplasts suggested that JcMADS05 is likely to act as a transcriptional activator, its function as a negative regulator of grain size could be mediated via activation of other repressors. In summary, the results provide a novel resource for future studies on MADS-box genes in physic nut, especially with respect to their effects on seed size.

Conclusions
In this study, we identified 63 JcMADS genes in the physic nut genome, and characterized their expression profiles under normal growth and abiotic stress conditions. Transgenic expression in rice of one of the genes (JcMADS05) reduced grain size, 1000-grain weight and yield per plant, supporting the hypothesis that some members of the family participate in the regulation of physic nut seed development. These findings provide insights facilitating prediction of the functions of MADS-box genes in stress tolerance and seed development, and comprehensive analysis of the gene family produced results that will be helpful in screening genes for further functional characterization and for the genetic improvement of agronomic traits in physic nut.

Plant materials
An inbred cultivar of J. curcas, GZQX0401, was used in this research, since its genome has been fully sequenced [25].

Conserved Motif And Chromosomal Distribution
The conserved motifs of individual MADS-box proteins were identified using the MEME server (http://alternate.meme-suite.org/). MEME was run with the following parameters: site distribution (Zero or one occurrence per sequence), motif number (20), motif width (between 6 and 100 wide). Chromosomal locations of JcMADS genes were obtained as described by Wu [25], and linkage maps of the JcMADS genes were drawn with the MapChart software package. To detected the transcript abundance of JcMADS genes in physic nut, root, leaf and stem cortex tissue from two-week-old seedlings, and seeds at day 14 (early developmental stage S1) and day 35 (filling and maturation stage S2) after pollination, were collected and stored in an ultra-low temperature freezer for further digital gene expression analysis.
Salinity and drought stresses were initiated at the six-leaf stage. For salinity stress, Hoagland solution containing 100 mM NaCl was used to irrigate seedlings daily. To apply drought stress, stop watering. Leaves were collected at 2 days, 4 days and 7 days after drought stress and 2 hours, 2 days and 4 days after salinity stress. Samples were immediately stored in an ultra-low temperature freezer for further digital gene expression and qRT-PCR analyses; raw sequence data were obtained based on standard protocols and submitted to the sequence read archive (SRA) at NCBI (accession nos. are PRJNA257901 (for the drought stress data) and PRJNA244896 (for the salt stress data)).

Subcellular Localization And Transcriptional Activation Analysis
The amplified coding region of JcMADS05 without the termination codon was inserted into

Gene Cloning And Plant Transformation
The coding sequence of JcMADS05 was amplified by RT-PCR from total RNA isolated from physic nut seed with the JcMADS05-F and JcMADS05-R primers given in Table S1, and cloned into the pMD18-T vector. Successful amplification of the target gene was confirmed by DNA sequencing. And then the target sequence was excised from the pMD18-T vector after digestion with Kpn I and Xba I, then cloned into the pCAMBIA1301 vector at the Kpn I/Xba I site under the control of the CaMV 35S promoter. The resulting constructs were introduced into Agrobacterium (strain EHA105) by the freeze-thaw procedure, and strains harboring the constructs were transformed into calli of rice cv. ZH11, according to the method of Tang      Patterns of expression of each JcMADS gene in physic nut roots, stem cortex, leaves, and seeds at an early developmental stage (S1) and filling stage (S2), with a colored scale indicating expression levels shown at the bottom.

Figure 5
Levels of expression of the 63 JcMADS genes in physic nut leaves exposed to drought and salinity stresses: log2 ratios of signals from treated versus control leaves are presented as a heat map based on transcriptomic data, with the color scale shown at the bottom. NA: not available.

Figure 6
The product of the JcMADS05 gene is localized in the nucleus. Scale bar, 10 μM.