Phylogenetic Relationships Of The Jcmads Proteins
To clarify the phylogenetic relationships of the physic nut MADS family proteins with the previously reported members of the family in Arabidopsis, an unrooted tree was constructed by MEGA6 using the neighbor-joining method (Fig. 1). On the basis of full length amino acid sequence conserved domain and similarity, we subdivided the 171 typical members of the MADS gene family into 5 groups (designated MIKC, Mα, Mβ, Mγ, Mδ), according to the previous classification of MADS proteins from Arabidopsis [9]. Of the 63 inferred physic nut JcMADS proteins, thirty-two were assigned to group MIKC (JcMADS03, 04, 05, 07, 12, 16, 17, 19, 20, 22, 24, 25, 26, 27, 28, 29, 31, 32, 37, 38, 42, 44, 46, 47, 49, 50, 52, 53, 54, 55, 56, 63), thirteen to group Mα (JcMADS01, 06, 08, 11, 13, 14, 33, 35, 39, 43, 57, 58, 59), four to group Mβ (JcMADS09, 48, 51, 60), six to group Mγ (JcMADS10, 15, 34, 36, 41, 62) and eight to group Mδ (JcMADS02, 18, 21, 23, 30, 40, 45, 61). In the phylogenetic tree, some members of the JcMADS gene family formed related sister pairs (Fig. 1): JcMADS11 and 35, 13 and 14, 02 and 45, 10 and 36, 07 and 55, 32 and 54. There were also triplets (JcMADS06, 57 and 58; 34, 41 and 62; 09, 51 and 60; 03, 04 and 05), and a set of quadruplets in the case of JcMADS01, 08, 33 and 39. The tree indicated that proteins in group MIKC were the most numerous; it contained 39 AtMADS and 32 JcMADS proteins
Conserved Motifs In Jcmads Proteins
The structures of proteins encoded by JcMADS genes were analyzed using the MEME online software tool. Twenty conserved motifs, which we named motifs 1–20, were identified in the 63 JcMADS proteins (Fig. 2 and Additional File 2). As expected, motif 1 and motif 4 corresponded to the typical MADS-box domain, and motif 1 was found in all the JcMADS proteins. Motif 9, specifying the K domain, was found in most MIKC type proteins; the exceptions were JcMADS17, 31, 37, 52, 56 and 63, which were relatively short amino acid sequences. In addition to these known functional motifs, some of unknown function were also detected. Examples included motifs 5, 10 and 20 (detected only in group Mγ), and motif 13 (found only in groups MIKC and Mβ). Motif 12 was found only in group MIKC, while motifs 14 and 19 was detected only in group Mδ. Additionally, most conserved motifs detected in JcMADS proteins were clade-specifically assigned in different groups, implying similarity of function within a given group.
Chromosomal localization of JcMADS genes
We mapped 62 of the 63 JcMADS genes (all except JcMADS63) to LGs based on a previously published report on the physic nut genome [25]. As shown in Fig. 3, we found that LGs 4 and 7 had more members of the JcMADS gene family than other LGs, with eleven and nine JcMADS genes respectively. They were followed by LGs 2, 3, 5 and 10, each of which had six JcMADS genes. In addition, there was five JcMADS genes on each of LGs 6 and 9, three on LG8, three on LG11 and two on LG1. The results also indicated that most JcMADS genes were on the lower and middle regions of the LGs. Tandem duplications, defined as tandem repeats which are separated by < 4 non-homologous spacers or are genes located within 50 kb of each other [26], were found among these members of the JcMADS gene family. The gene pairs present as tandem repeats (T) were T1 (JcMADS03 and 04), T2 (JcMADS06 and 07), T3 (JcMADS12, 13 and 14), T4 (JcMADS19 and 20), T5 (JcMADS26 and 27), T6 (JcMADS30 and 31), T7 (JcMADS33 and 34), T8 (JcMADS37 and 38), T9 (JcMADS43 and 44), T10 (JcMADS46 and 47), T11 (JcMADS50, 51 and 52) and T12 (JcMADS56, 57 and 58), on LG2, LG2, LG3, LG4, LG5, LG5, LG6, LG7, LG7, LG8, LG9 and LG10 respectively.
Expression profile of JcMADS genes under non-stressed growth condition
To clarify the roles of the JcMADS in regulating physic nut development, we examined the expression profiles of JcMADS genes in roots, stem cortex, leaves, and seeds (S1 and S2) under non-stressed growth conditions based on data from RNA sequencing (RNA-seq) (Additional File 3 and Fig. 4). The result suggested that fifty of the predicted JcMADS genes were expressed in at least one of the organs examined, while thirteen (JcMADS06, 13, 17, 35, 39, 40, 49, 52, 57, 58, 60, 61 and 63) were not expressed in any of these tissues. Of the 50 JcMADS genes for which expression was detected, two (JcMADS09 and 47) were highly expressed across all the tissues sampled, ten (JcMADS03, 05, 11, 12, 14, 23, 34, 37, 46 and 62) were expressed only in seeds, thirteen (JcMADS04, 15, 21, 24, 25, 29, 38, 41, 43, 48, 50, 51 and 54) exhibited highest expression in seeds, four (JcMADS08, 16, 42 and 44) preferred to be expressed in roots, and one (JcMADS28) was most strongly expressed in the stem cortex.
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.
Expression profile of JcMADS under abiotic stress conditions
Many studies have suggested that some MADS-box genes encode proteins involved in the regulation of abiotic stresss [21, 27–28]. We therefore further investigated the patterns of expression of JcMADS genes in leaves after 2 d, 4 d and 7 d of drought stress and after 2 h, 2 d and 4 d of salinity stress according to data from RNA-sEq. As shown in Fig. 5, the transcript abundances of seven JcMADS genes indicated at least a twofold enhancement or reduction compared with the control in response to at least one stress at one or more time points. Of these seven genes detected as having differential expression, three (JcMADS42, 43 and 47) exhibited significantly induced or inhibited expression in response to drought and salinity stresses, three (JcMADS22, 30 and 53) showed differential expression only in response to drought stress, and JcMADS15 responded solely to salt stress.
JcMADS05 is a nucleus-localized transcriptional activator
To confirm the subcellular localization of the protein encoded by JcMADS05 gene, the 35S:JcMADS05-YFP fusion construct and the 35S:YFP empty vector were introduced into Arabidopsis protoplasts. The fluorescence signals from the protoplasts were then observed immediately by confocal laser-scanning microscopy. As shown in Fig. 6, we observed that the yellow fluorescent signal was distributed throughout the whole of the cell when the 35S:YFP vector was used, whereas in protoplasts harboring the construct 35S:JcMADS05-YFP a strong yellow fluorescent signal was detected in the nuclei. These findings indicate that JcMADS05 gene is located in the nucleus.
A dual-luciferase assay was used to examine the transcription activation activity of JcMADS05 protein. The full-length CDS of JcMADS05 was attached to the vector pBD, then the pBD-JcMADS05 fusion effector vector and the p5 × GAL-Reporter vector were transformed into Arabidopsis protoplasts. The results indicated that the LUC/REN ratio was significantly lower in the control protoplasts (pBD) than in the pBD-JcMADS05 group. Our data suggest that the full-length JcMADS05 has transactivation activity (Fig. 7). Based on the above results, we drew the conclusion that JcMADS05 functions as a transcription activator.
Phenotypic analysis of transgenic rice plants expressing JcMADS05
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.
Overexpression of JcMADS05 reduces the grain size in transgenic rice
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.