Genome-wide identification of ZjMAPKKKs
A total of 56 ZjMAPKKKs were defined and all of them have the key S-TKc (serine/threonine protein kinase) domain and other conserved protein kinase domains (Additional file. 2). With the purpose of clearly understanding and discrimination of the MAPKKK genes, the Locus of MAPKKKs to ZjMAPKKKs according to the nomenclature suggestions of Arabidopsis was designated as, of which Zj is short for Ziziphus jujuba and the series number of ZjMAPKKK1-56 was coded in term of their locations on chromosomes (Table 1). The ZjMAPKKKs distributed all over the 12 pseudo-chromosomes, excepted ZjMAPKKK44-56 could not match to corresponding chromosome.
Specific information of each CDS and amino acid sequences of ZjMAPKKKs were listed in the Additional file 1 and 7. In addition, according to the specific conserved signature motif, all the ZjMAPKKKs could be divided into two subfamilies (Raf and MEKK); no ZIK subfamily members could be identified. Furthermore, as shown in Table 1, the length of the CDS sequence ranged from 762 bp (ZjMAPKKK36) to 4455 bp (ZjMAPKKK7), with an average length of 1804 bp. The amino acid sequence length of ZjMAPKKKs varied from 253 (ZjMAPKKK36) to 1484 (ZjMAPKKK7) amino acids (aa); average length was 600 aa. The predicted molecular weight (Mw) of these proteins ranged from 28.29 (ZjMAPKKK36) to 160.66 (ZjMAPKKK7) and the theoretical isoelectric point (pI) ranged from 4.78 to 9.34, respectively.
Phylogenetic analysis of ZjMAPKKKs genes
In order to assess the phylogenetic relationships between Chinese jujube and Arobidopsis, the phylogenetic tree was constructed with the total 136 protein sequences (56 ZjMAPKKKs and 80 AtMAPKKKs). As illustrated in Fig. 1, the members of AtMAPKKKs could be clustered into three categories, Raf, ZIK and MEKK, indicating that the method for the phylogenetic tree was reliable. However, the 56 members of ZjMAPKKKs could only be clustered into two subfamilies, Raf and MEKK. In addition, the largest Raf subfamily consisted of 41 members and the other 15 members of ZjMAPKKKs belonged to MEKK subfamily, and none of them could be defined into ZIK subfamily. Morever, some ZjMAPKKKs located on the same chromosome, shown little divergence, but clustered into the same group, such as ZjMAPKKK36, 37 and 40, ZjMAPKKK15 and 16, ZjMAPKKK38 and 39. These results indicated that some duplication of ZjMAPKKKs took place during the evolutionary process of jujube.
Conserved domains and gene structure analysis of ZjMAPKKKs
Within the analysis of MEME software, five main conserved motifs were identified in all 56 ZjMAPKKKs (Fig.2). The motif 1, 3 and 4 were founded in all the ZjMAPKKKs, while the other two motifs were observed in all the members of Raf subfamily. Additionally, the members of MEKK subfamily could be divided into two groups, one group contains motifs 1-4, including ZjMAPKKK21, 56, 6, 31, 10 and 25. The remaining members only consisted of motifs 1, 3 and 4. These results illustrated that the ZjMAPKKKs share the same conserved motifs which further indicate that the protein structures for each subfamily were highly conserved.
For the analysis of the contents of exon/introns, the difference among ZjMAPKKKs was significant. As shown in Fig. 3 and Additional file 8, the number of exons in ZjMAPKKKs ranged from 1 (ZjMAPKKK9, 12, 29, 35, 36, 44 and 54) to 19 (ZjMAPKKK42). Interestingly, the members of ZjMAPKKKs which only contained 1 exon, all belong to MEKK subfamily (47%), but the highest number of exons in this subfamily could arrive to 17 (ZjMAPKKK25 and 10), the average number was 5.6. This demonstrated that more significant loss and gain of exon took place in this subfamily during the process of evolution. For the subfamily of Raf, the number of exon varied from 2 (ZjMAPKKK16 and 28) to 19 (ZjMAPKKK42), with the average number of 9.56. Even the significant variation of the number of exons existed in the Raf and MEKK subfamilies, some exon structure patterns were clearly conserved in close paralogs. For instance, the ZjMAPKKK24 and 49 have 12 exons, ZjMAPKKK37 and 40 have 2 exons, and they were all closely clustered in the same phylogenetic tree. Collectively, the evolutionary different organization of ZjMAPKKKs gene structures between Raf and MEKK subfamilies indicated that the tandem and segmental duplication events might occur in the ancient time and the diverse exon structures might function differently in the jujube genome.
Furthermore, with the multiple protein alignment of ZjMAPKKKs, the Raf-specific signature motif: GTXX(W/Y)MAPE was founded in the Raf subfamily and the kinase domain located at the N terminal or C terminal. In contrast, the less conserved MEKK-specific signature motif: G(T/S)PX(W/F)MAPEV was observed in MEKK subfamily, while the kinase domain located at three positions: N- or C-terminal or the central part of the proteins (Fig.4). The features of the signature motifs of ZjMAPKKKs were consistent with other orthologues in other plants that fulfill them the important role in diverse signal transduction in plants.
Synteny analysis of ZjMAPKKK genes
Furthermore, a tandem duplication event was firstly analyzed according to the principle that two or more genes located on a chromosomal region within 200 kb [34]. As shown in fig.5, One pair of ZjMAPKKKs (15/16) was the only tandem duplication event on LG5. In addition, 13 segmental duplication events with 22 ZjMAPKKKs were also identified. These results indicated that some ZjMAPKKKs were possibly generated by gene duplication and the segmental duplication events played a major driving force for ZjMAPKKKs evolution.
Phytoplasma detection in different phytoplasma infected tissues
In order to get insight to understand the function of ZjMAPKKKs involved in phytoplasma infection, the expression level of individual ZjMAPKKKs was detected by qPCR in two kind of plant materials one material was infected by phytoplasma with three different symptoms, including witches’ broom leaves, phyllody leaves and apparent normal leaves from diseased plants (in vivo) and the other plant material was sterile cultivated tissues of JWB plantlets (in vitro). Firstly, the phytoplasma concentration in in the first plant material with three sympotems has been detected by Xue et al. (2018) [21], and the phytoplasma determination in the sterile cultivated tissues of JWB plantlets shown that the fluorescent spots formed a large bright circle in the petiole phloem (Additional file 5). These results guaranteed the following test on the function of ZjMAPKKKs in response to phytoplasma infection.
Expression analysis of ZjMAPKKKs in witches’ broom leaves
As shown in Additional file 9 and Fig. 6 (A), the heat map showed the expression levels of ZjMAPKKKs with significantly different patterns in witches’ broom leaves from June to September. 42 candidates expressing level could be detectable, but the expression levels of the other 14 ZjMAPKKKs were very low and could not be detectable. Finally these undetectable expressing ZjMAPKKKs genes were considered as the redundant candidates in our research and were not selected for further calculation and analysis. Among them, the most significant transcript induction took place at the early stage (June or July) when the witches’ broom began to grow. For instance, the ZjMAPKKK13, 14, 15, 23, 34,42, 44, 47 and 56 were highly induced in June or July (above 2-folds), afterwards, began to decrease from August to September as shown in Fig. 6 (B). However, the ZjMAPKKK3, 43 and 50 were down-expressed from June to September. In addition, two members of ZjMAPKKKs (26 and 45) remained high level expression constantly from June to September, indicating these two candidates might be the potentially key MAPKKKs in response to phytoplasma infection. However, it should be kept in mind that the clustering of the expression profiles of ZjMAPKKKs was not consistent with the gene similarities, illustrating the gene function did not only rely on the gene structure.
Expression analysis of ZjMAPKKKs in phyllody leaves
As depicted above, the transcript abundance of ZjMAPKKKs were as well as investigated in the tissue of phyllody leaves. The heat map of the expressing ZjMAPKKKs was indicated in Fig. 7 (A). Several of the ZjMAPKKKs were highly expressed in June or July and then the expression levels of them decreased from August to September, while most of the ZjMAPKKKs showed no significant changes or down regulated. For the details of each single ZjMAPKKKs expressing level could be seen in the Fig. 7 (B), the ZjMAPKKK10, 14, 15 34, 44 and 56 were significantly up regulated at the early stage (June or July). However,ten of the ZjMAPKKKs showed significantly down regulated, including ZjMAPKKK3, 16, 18, 41, 43, 50, 51, 52, 53 and 55. As same as the expressing pattern of ZjMAPKKK26 and 45 in the tissue of witches’ broom leaves, these two members were also highly up regulated from June to September in the tissue of phyllody leaves.
Expression analysis of ZjMAPKKKs in apparent normal leaves
Furthermore, the apparent normal leaves that were infected by phytoplasma but shown no apparent symptoms was used to test which ZjMAPKKKs really play roles in response to phytoplasma infection in the non-strong disease leaves . Interestingly, the heat map figure showed different expressing pattern of ZjMAPKKKs in the tissue of apparent normal leaves (Fig. 8B). Seldom genes were highly up regulated and most of them showed down regulated pattern. For example, the ZjMAPKKK1, 3, 7, 16, 17, 18, 19, 41, 43, 50, 51 and 53 were down regulated from June to September, while the ZjMAPKKK28, 34 and 47 were significantly up regulated in June or July, and the ZjMAPKKK27 and 54 were up regulated from August or September. However, ZjMAPKKK26 and 45 showed the same high expressed pattern in the tissue of apparent normal leaves from June to September (Fig. 8 A).
To sum up, in the above-mentioned three phytoplasma infected tissues from four different time series, ZjMAPKKK26 was significantly up regulated (8 times) and the ZjMAPKKK45 were highly induced 10 times. Moreover, with the infecting development of phytoplasmas, the symptoms of which observed on jujube leaves were gradually stronger from the apparent normal leaves, phyllody leaves to withches’ broom leaves [21]. For the analysis of this aspect, the expression level of ZjMAPKKK26 was highly induced in phyllody leaves (~6 fold) in June, but not within other two symptom leaves. Then with time and infecting development, ZjMAPKKK26 was up regulated in witches’ broom leaves (~3 fold) in July and down regulated in phyllody leaves (~2 fold) in the meantime. However, ZjMAPKKK45 was highly induced in apparent normal leaves and phyllody leaves (~3 and ~6 fold, respectively) in June and began to be down regulated from July to September, but it was also induced in witches’ broom leaves constantly (~2 fold) (Fig. 6, 7 and 8). These results demonstrated within the infection of phytoplasmas, the ZjMAPKKK26 was quickly response in phyllody leaves and then highly induced in witches’ broom leaves, however, ZjMAPKKK45 response faster than ZjMAPKKK26 because of its high expression in apparent leaves in June. In contrast to these two up regulated ZjMAPKKK genes, theZjMAPKKK3, 43 and 50 were down regulated 9, 9, 10 times, respectively.
Expression analysis of ZjMAPKKKs in the sterile cultivated tissues of JWB plantlets
After investigating the expression profiles of ZjMAPKKKs in field tissues, we further detected their expression levels in the sterile cultivated (in vitro) tissues of JWB plantlets and the healthy plantlets were used as control. As shown in Fig. 9, the expressing profiles of ZjMAPKKKs indicated significant disparities compared with the above results. Only four members of ZjMAPKKKs were significantly induced in the disease plants, i.e., 4, 10, 25 and 44. While the ZjMAPKKK7, 30, 35, 37, 40, 41, 43 and 46 were significantly down regulated. The others showed none significantly changes.