The Mitogen-activated protein kinase (MAPK) cascade is a conserved signaling transduction pathway, which plays important roles in the regulation of the cell cycle, plant growth and development, and responses to hormones and environmental stresses   . The core MAPK cascade pathway consists of three proteins kinases: MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK) and MAPK, which phosphorylates and activates the latter one in a specific way  . Besides, the fourth level of kinases, MAPKKK kinases (MAPKKKKs), may function as adapters linking upstream signals to the core MAPK cascade pathways  . In the process, a variety of external stress signals are cascaded, amplified and are transmitted into the cell, which subsequently activates multiple downstream regulatory factors.
The MAPKs can be categorised into four subfamilies (A - D groups) based on amino acid sequences similarities. Groups A, B and C possess a common phosphorylation motif TEY in their activation loop, whereas group D MAPKs display TDY motif instead of TEY motif   . Besides, the four groups possess diverse CD domain in their C-terminal extension, which functions as a docking site for MAPKKs, phosphatases and protein substrates  . CD domain in group A and B MAPKs possesses common sequence characteristics [LH][LHY]Dxx[DE]xx[DE]EPxC (where x represents any amino acid) that includes two adjacent acidic residues (D and E)  , whereas group C MAPKs show modified sequence features in the hydrophobic residues  , and the CD domain is not found in the Group D MAPK sequences  .
Group C MAPKs contain 4 members, MPK1, MPK2, MPK7 and MPK14. The function studies of group C MAPKs appeared in the recent 10 years, which lags behind group A and B. Studies reveals that group C MAPKs are involved in various signaling processes and have important biological functions in the response of stress resistance and the regulation of plant growth and development. The transcriptional expression of Arabidopsis MPK1 and MPK2 and pea PsMPK2 are induced by wounding, JA, abscisic acid, and H2O2   . The ZmMPK7 in maize is transcriptionally induced by ABA and H2O2  . The expression of GhMPK7 in cotton is induced by chemical, biotic and abiotic inducers, such as NaCl, wounding, pathogen, SA, MeJA and H2O2, and genetic evidence showed that GhMPK7 might play an important role in SA-regulated broad-spectrum resistance to pathogen infection as well as in regulation of plant growth and development  . The transcription of the SlMPK7 in tomato leaves was triggered by hydrogen peroxide (H2O2) anabiotic stresses and exogenous signaling molecules. Moreover, overexpression of SlMPK7 enhanced tolerance to chilling stress in tomato  . Tomato MPK1 and MPK2 are involved in plant disease resistance and abiotic stresses, and MPK2 played a more critical role than MPK1 in 24-epibrassinolide (EBR)-induced apoplastic H2O2 accumulation  . Studies showed that the Arabidopsis four group C MAPKs are activated by upstream MKK3, and MKK3-MPK7 is involved in pathogen signaling  . Besides, the ABA core machinery is required for the activation of the MAP3K17/18-MKK3-MPK1/2/7/14  .
Populus trichocarpa is an important genus of tree that plays a vital role in economic and ecosystem services all over the world. Environmental biotic and abiotic stresses could have harmful effects on the development and growth of poplars. Therefore, the insights into the molecular mechanisms of environmental adaptability of poplar are highly regarded. It has been reported that the genome of Populus trichocarpa encodes 21 MAPKs (PtMPK), and these MAPKs are divided into A-D groups   . The group C MAPKs consist of PtMPK1, PtMPK2, PtMPK7 and PtMPK14   . However, the biological function of these MAPKs is completely unknown.
Poplar 84K (Populus alba × P. glandulosa cv. “84K”) is a new hybrid variety. Poplar 84K is easy to grow roots and grows fast in young seedling stage. Besides, it has advantage of good material, strong resistance and wide adaptability, and is an important fast-growing timber species  . In this study, a group C MAPK gene, 84KMPK14 was cloned from hybrid poplar 84K. The expression pattern of 84KMPK14 gene in plant tissues was analyzed using quantitative realtime RT-PCR (qRT-PCR). The results indicated that 84KMPK14 might have different developmental function with PtMPK14 in other poplar species Populus trichocarpa. Moreover, the expression response analysis of 84KMPK14 gene to low temperature, high temperature, high salinity, drought and exogenous H2O2 revealed that 84KMPK14 is regulated by H2O2 and might be a candidate gene underlying these abiotic stresses. This study is meaningful to reveal that 84KMPK14 might be an important regulator in development and abiotic stress response in 84K poplar.
2. Materials and Methods
The experiment was conducted at Shandong Agricultural University and the Flower Institute of Forestry College.
2.1. Plant Materials
The 84K poplar plantlets transformation was set up by the Flower Institute of Forestry College. The 84K poplar plantlets were propagated by tissue culture. The 2 cm shoot tips were cut and cultured in sterilizing containers containing basal MS medium supplemented with 0.01 mg/L NAA and 0.1 mg/L IBA, at 25˚C/25˚C under 16 h light/8 h dark photoperiod and 12,000 lux light intensity. After 4 weeks, the seedlings were transferred to polystyrene trays containing Hoagland’s solution.
Two week-old hydroponic polar plantles were submerged to various treatments. For stress treatments, 200 mM NaCl, 10% PEG 6000 and 50 mM H2O2 was applied in the Hoagland’s nutrient solution to examine adaptive responses to NaCl, dought and H2O2, and the seedlings were placed in a 4˚C or 42˚C growth chamber to examine adaptive responses to cold or heat stress, at the same time, seedlings in the basic nutrient solution under normal condition were used as mock control. Shoot apexs were collected directly into liquid nitrogen after 0 h, 1 h, 3 h, 6 h and 12 h of all the treatments and stored in a −80˚C freezer.
2.2. RNA Extraction and cDNA Synthesis
The total RNA was isolated and was treated with Recombinant DNaseI (RNase-free) with TRNzol reagent (TransGen Biotech, China) according to the manufacturer’s protocol. Then the first-strand cDNA was synthesized from 2 μg total RNA using M-MLV reverse transcriptase and oligo (dT) 15 as primer with the PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, China).
2.3. PCR Cloning of 84KMPK14 Gene
The full-length cDNA of 84KMPK14 was amplified from the cDNAs by using the following primer pairs: 84KMPK14-F (5'-GAAATGGCAACTTTAGTGGAGC-3') and 84KMPK14-R (5'-GCCGGAGTTTCATTGATATTGC-3'). The PCR amplification was performed as follows: the reaction mixture was first pre-denatured at 94˚C for 3 min; then at 94˚C for 15 s, 58˚C for 15 s, and 72˚C for 2 min for a total of 30 cycles; followed by 72˚C for 10 min. The PCR products were detected with 1% agarose gel electrophoresis. The target PCR fragment was recovered with the Nucleo Trap Gel Extraction Kit (TaKaRa) and was ligated to the Blunt3 vector and then was transformed into Escherichia coli DH5α. The positive clones were selected and identified by sequencing.
2.4. Bioinformatics Analysis
The BLASTP program on the National Center for Biotechnology Information was used to predict the conservative domain of 84KMPK14 protein. BLASTX (NCBI) was used to study the homology of the nucleotide sequences. DNAMAN was used to conduct multiple sequence alignment. Phylogenetic tree was constructed by MEGA5.0 software according to the neighbor-joining method, and tested by bootstrap, which was repeated 1000 times. Furthermore, the protParam tool was used to predict the physicochemical properties of 84KMPK14 protein and ExPaSy-SOPMA was used to predict protein secondary structure.
2.5. Real-Time Quantitative PCR Analysis
The qRT-PCR reactions were performed on 2 μL of each cDNA dilution using the SYBR® Premix ExTaq (TaKaRa) and CFX96TM Real-time PCR Detection System (Bio-Rad, America) according to the manufacturer’s instructions. The reaction procedure was as follows: started with an initial step of 95˚C for 30 s; 39 cycles at 95˚C for 30 s, 60˚C for 30 s to calculate cycle threshold values; finally 60˚C for 30 s and 95˚C for 30 s to obtain the melting curve. The samples from each biological replicate were assayed in triplicate and included three negative controls using water instead of cDNA. The Actin gene (POPTR_0001s31700g) was used as the reference gene. The specific primers used for quantitative analysis was designed using the Primer 5.0 software. The primers in qPCR were used as follows: 84KMPK14q-F (5'-CCTTATGCAAGGAGAATCCATTT-3'), 84KMPK14q-R (5'-GCTTCTGTCACACTAATTCTCTTAG-3'), Actin-F (5'-TTCTACAAGTGCTTTGATGGTGAGTTC-3'), Actin-R (5'-CTATTCGATACATAGAAGATCAGAATGTTC-3'). Relative transcript levels were calculated using the comparative Ct method and results were normalized to the Actin gene.
3.1. Cloning and Sequence Analysis of 84KMPK14 Gene
To study the roles of group C MAPKs in poplar, we cloned the candidate gene homologous to MPK14 gene from young leaves of 84K poplar by RT-PCR (Figure 1), and it was designated as 84KMPK14.
The full-length cDNA of 84KMPK14 consisted of 1107 nucleotides, encoding a polypeptide of 369 amino acids and having the isoelectric point of 7.66 and the relative molecular weight of 42,693.09 Da. The instability index is computed to be 35.04 and the grand average of hydropathicity was −0.222. The phosphorylation site prediction results demonstrated that the 84KMPK14 protein sequence possesses 16 serine (Ser) phosphorylation sites, 15 threonine (Thr) phosphorylation sites, and 16 tyrosine phosphorylation sites. Protein sequence and structural analysis showed that 84KMPK14 contains a conserved-TEY-signature was found in its activation loop (Figure 2). Moreover, a variant conserved common docking (CD) domain was found in the C-terminal part of the protein (Figure 2, Figure 3).
Phylogenetic tree of plant group C MAPKs was constructed by using the full-length protein sequences (Figure 4). The dendrogram showed that the 84KMPK14 protein was most closely related to Populus trichocarpa PtMPK7 and PtMPK14, Arabidopsis thaliana AtMPK7 and AtMPK14 (Figure 4). Amino acid sequence identity analysis indicated that 84KMPK14 protein was highly homologous to MPK14 in Populus trichocarpa (PtMPK14; XP002310268.1), with 96.75% identity. Thus, it is most likely that the 84KMPK14 encodes a member of a putative group C MPK14 in 84K poplar, and it was renamed as 84KMPK14.
Figure 1. PCR products of 84KMPK14 full-length cDNA. M, DNA Marker DL2000.
Figure 2. The protein structural organization of 84KMPK14. Polypeptide subtrate binding site TEY motif CD domain A-loop (activation loop) and the conserved functional domains family-specific protein signatures for 84KMPK14.
Figure 3. Alignment of amino acid sequences of 84KMPK14 with group C MAPKs in Populus trichocarpa and Arabidopsis thaliana. Identical amino acid residues are shaded in black, and similar amino acid residues are shaded in gray. TEY, TEY phosphorylation moif; CD domain, atypical conserved common docking domain.
Figure 4. The phylogenetic relationship of 84KMPK14 with other plant MAPKs.
3.2. Expression Profile of 84KMPK14 Gene
To understand the expression pattern of 84KMPK14 gene, qRT-PCR was performed to detect the transcripts in the tissues of 84K poplar. The tissues included young leaves, mature leaves, shoot apex, upper stems, lower stems, and roots. As shown in Figure 5, the 84KMPK14 gene transcripts were detected in all of the examined tissues, and were found to be at relatively higher levels in young leaves and roots compared with other tissues. The expression level of 84KMPK14 in young leaves is about 3.39 times of that in mature leaves, in which the gene exhibit the lowest expression level. However, the transcripts accumulated in shoot apex, upper stems and lower stems were relatively uniform. These results implied that the 84KMPK14 gene is not mainly expressed in young tissues.
3.3. Expression of 84KMPK14 in Response to Abiotic Stress
Abiotic stresses such as low temperature, high temperature, drought and high salinity are common unfavorable environmental conditions, which the plants are frequently subjected to. To investigate whether the 84KMPK14 gene responses to abiotic stresses, the expression changes in response to a time course 4˚C, 42˚C, PEG and NaCl stress treatments were analyzed using qRT-PCR technique. To exclude other effect such as temperature and light on the gene expression, a time course H2O2 treatment in normal condition was used as control (mock treatment). The results showed that the relative expression of 84KMPK14 in normal physiological state significantly unstable with time during the mock treatment (Figure 6).
As shown in Figure 6, the 84KMPK14 gene showed remarkably expression response to different treatment compared with the corresponding mock control. After 4˚C treatment, the transcript level of the 84KMPK14 gene significantly decreased after 1 h treatment, and the minimum expression level appeared at 3 h treatment (Figure 6(a)). The expression level of 84KMPK14 under 42˚C stress
Figure 5. Expression pattern analysis of the 84KMPK14 gene in various tissues. YL, young leaves; ML, mature leaves; SA, shoot apex; US, upper stems; LS, lower stems; R, roots.
(a) (b) (c) (d) (e)
Figure 6. The expression responses of 84KMPK14 gene to abiotic stress and H2O2 treatment in 84K poplar. All treatments were repeated three times with similar results.
exhibited down-regulated expression only at 3 h treatment, and the expression decreased to 54% of the mock control (Figure 6(b)). When treated with NaCl, the transcripts of 84KMPK14 remarkablely decreased at 1 h (35% of the mock control), and then significantly increased and reached to the maximum level at 3 h (2.13-fold of the mock control), and subsequently returned to the mock control level at 6 h (Figure 6(c)). After PEG stress, the expression of 84KMPK14 significantly decreased at 1 h treatment, then reverted to the mock level at 3 h treatment, and then, the expression was up-regulate, and reached to the maximum at 12 h (2.33-fold of the mock control) (Figure 6(d)). This findings indicate that the poplar 84KMPK14 gene response to 4˚C, 42˚C, NaCl and PEG stress in transcriptional level, and might be a candidate gene underlying these tolerance.
3.4. Expression of 84KMPK14 in Response to H2O2
Aerobic metabolisms in plant cells continuously produce reactive oxygen species (ROS), and it is in equilibrium in normal conditions. Whereas, biotic stress and abiotic stress could disturbing this balance and exacerbate ROS generation, and as a result, it finally causes oxidative stress in plants  . To investigate whether 84KMPK14 gene response to oxidative stress, 84K poplars were treated with 50 mM exogenous H2O2, then choose shoot apex and used the qRT-PCR to analyze the expression changes in response to a time-course H2O2 treatment. The results are shown in Figure 6(e). The expression of 84KMPK14 was strong induced at 1 h H2O2 treatment, which displayed 12.00-fold of that in mock control. After 3 h of exposure to H2O2, the induced expression of 84KMPK14 gene decreased to 3.12-fold compared with that in the mock control, and then tended to be increased later. These results prove that the expression of 84KMPK14 is regulated by H2O2.
Poplar is one of the main forestation species and commercial tree in the world. The environmental adaptability of poplar is closely related to the timber production. MAPK cascade is a common mode of signal transduction, which can deliver external stimuli to the cells and induce corresponding physiological responses. MAPK gene families in plants are divided into A-D types, and MPK14 belongs to C group   . In this study, we cloned a new C group MAPK gene, MPK14 from 84K poplar. The amino acid sequence alignment showed that MPK14 contains typical serine/threonine protein kinase domain and a conserved-TEY-signature in its activation loop (Figure 2, Figure 4), which is highly conserved in C group MAPKs in plants. The CD domain in MAPKs are known as a docking site for MAPKKs, phosphatases, and protein substrates  . Meanwhile, the corresponding modified CD domains in group C MPKs is considered as the probable specific interaction site with MKK3 in Arabidopsis  . In this study, we found that there are 4 modified residues in the CD domains of 84KMPK14 compared with that in PtMPK14, indicating that the specific interaction of MPK14 with MAPKK in the two poplar tree might be discrepant, and it is to be demonstrated by further experiments.
The previous study  proposed that the transgenic Nicotina benthamiana overexpressing GhMPK7 showed obvious phenotypic alterations that germinated earlier and grew faster than the wild-type plants, and these proved that C group MAPKs play an important role in regulating plant growth and development. In this study, the organ-specific expression profile analysis showed that the expression of 84KMPK14 was the highest in roots compared with other organs detected in this study (Figure 5). This is consist with PtMPK14 reported previously  , and we consider that MPK14 in poplar might have important biological roles in roots. The expression of 84KMPK14 was almost preferentially expressed in young tissues (Figure 5), indicating that 84KMPK14 might be involved in the growth and development of poplar. Interestingly, except for roots, we found that the transcription level of 84KMPK14 was in the order of young leaves > shoot apex > upper stems, while the expression of PtMPK14 in these organs of Populus trichocarpa was the exact opposite  , so, we speculate that an discrepancy might be exist in the developmental function of MPK14 between the two poplar species. However, the function of 84KMPK14 in poplar growth and development remains to be further investigated.
When carrying out the stress response experiments, the expression of 84KMPK14 in 84K seedlings incubated under normal conditions was taken into consideration. The result showed that the transcription of 84KMPK14 is unstable during the processing time (1 - 12 h) (Figure 5), suggesting a rhythmic expression of 84KMPK14 might be existed. Rhythmic expression of Arabidopsis MPK7 has been reported On the contrary, no rhythmic expression of ZmMPK7 in maize was also been attested  . These results give a hint that rhythmic response of group C MAPK genes is differentiated across species and homologue genes.
Several studies have reported that the group C MAPKs response to abiotic stress in several plants  -   . In this study, the transcription kinetics analysis of 84KMPK14 under environmental stress showed that 84KMPK14 could be inhibited by low/high temperature, while the transcription level of 84KMPK14 dropped first and rose later after drought and high salinity treatment (Figure 6). To our interest, different responsive modes to several abiotic stress of group C MAPKs were reported. For example, OsMPK14 could be induced by salt, and be inhibited by drought in rice, but low temperature could only activate its expression after 6 h treatment  ; ZmMPK7 was detected no obviously response to PEG or 4˚C in maize seedlings  ; moreover, GhMPK7 accumulated after NaCl treatment, but it showed a negligible response to low/high temperature and PEG  . Thus, we deduce that the specific abiotic stress response of group C MAPK genes is not ubiquitous across homologues though they share high sequence similarity, and the abiotic stress responses between orthologs of MPK14 are differentiated at least between monocotyledonous and dicotyledonous plants.
Abiotic stress processes, such as drought, heat, salinity, are often associated with the production of ROS, which primarily act as signal transduction molecules in the abiotic stress-response signal transduction network, and then are also toxic byproducts of stress metabolism, which causes oxidative stress leading to cellular damage and ultimately cell death   . To date, studies have shown that several group A and B MAPKs are involved in the cross-talk between stresses and hormones  . In addition to this, group C MAPKs are also reported to be associated with oxidative stress. The induced accumulation expression of ZmMPK7 and GhMPK7 was also detected in response to H2O2   , which is consistent with our results (Figure 6). Genetic studies had proved the evidence that tomato MPK1/2 may be involved in EBR-induced apoplastic H2O2  . NADPH oxidase-produced H2O2 regulated MPK1/2 activation during acclimation apoplastic H2O2 and RBOH1-mediated MPK1/2 activation plays a critical role in induction of cross-acclimation to abiotic stresses in tomato plants  . In addition, ZmMPK7 in maize is responsible for the removal of reactive oxygen species  . Therefore, it is reasonable to speculate that 84KMPK14 might also be involved in the oxidative tolerance, and this might be a way by which 84KMPK14 participated in several abiotic stress resistance in 84K poplar.But, the mechanism of 84KMPK14 in response to abiotic and oxidative tolerance in poplar needs to be further studied through transgenic technology.
84K poplar is a variety of white poplar and it has the advantages of easy to grow roots, fast growing in seedling and young tree stage, good material quality and wide adaptability. Thus, it is valuable to study the adaptable biological mechanism in 84K poplar. Based on the discussion above, the biological function of 84KMPK14 in 84K poplar is worthy to be explored in depth through genetics means.
This study was supported by the National Natural Science Foundation of China (No. 31770706, 31601732).
*These authors contribute equally. #Corresponding authors.