RhMKK9, a rose MAP KINASE KINASE gene, is involved in rehydration-triggered ethylene production in rose gynoecia
- Jiwei Chen†1, 2,
- Qian Zhang†1, 2,
- Qigang Wang3,
- Ming Feng1, 2,
- Yang Li1, 2,
- Yonglu Meng1, 4,
- Yi Zhang1, 2,
- Guoqin Liu1, 2,
- Zhimin Ma1, 2,
- Hongzhi Wu5,
- Junping Gao1, 2 and
- Nan Ma1, 2Email author
© The Author(s). 2017
Received: 29 January 2016
Accepted: 9 February 2017
Published: 23 February 2017
Flower opening is an important process in the life cycle of flowering plants and is influenced by various endogenous and environmental factors. Our previous work demonstrated that rose (Rosa hybrida) flowers are highly sensitive to dehydration during flower opening and the water recovery process after dehydration induced ethylene production rapidly in flower gynoecia. In addition, this temporal- and spatial-specific ethylene production is attributed to a transient but robust activation of the rose MAP KINASE6-ACC SYNTHASE1 (RhMPK6-RhACS1) cascade in gynoecia. However, the upstream component of RhMPK6-RhACS1 is unknown, although RhMKK9 (MAP KINASE KINASE9), a rose homologue of Arabidopsis MKK9, could activate RhMPK6 in vitro. In this study, we monitored RhMKK2/4/5/9 expression, the potential upstream kinase to RhMPK6, in rose gynoecia during dehydration and rehydration.
We found only RhMKK9 was rapidly and strongly induced by rehydration. Silencing of RhMKK9 significantly decreased rehydration-triggered ethylene production. Consistently, the expression of several ethylene-responsive genes was down regulated in the petals of RhMKK9-silenced flowers. Moreover, we detected the DNA methylation level in the promoter and gene body of RhMKK9 by Chop-PCR. The results showed that rehydration specifically elevated the DNA methylation level on the RhMKK9 gene body, whereas it resulted in hypomethylation in its promoter.
Our results showed that RhMKK9 possibly acts as the upstream component of the RhMKK9-RhMPK6-RhACS1 cascade and is responsible for water recovery-triggered ethylene production in rose gynoecia, and epigenetic DNA methylation is involved in the regulation of RhMKK9 expression by rehydration.
KeywordsRose flower Gynoecia RhMKK9 Rehydration Ethylene biosynthesis DNA methylation
Plants are exposed to various abiotic and biotic stresses because of their sessile life style. Therefore, for improved survival, plants develop a system that can rapidly sense signals from a changing environment and respond adaptively and/or defensively by modulating the internal physiological, biochemical, and molecular processes [1, 2]. For most crops, water deficit causes a major limitation to the yield. It has been well documented that water deficit results in several physiological changes, including wilting, stomatal closure, hormone imbalance, and suppression of cell growth and photosynthesis [3, 4]. After dehydration, plants are able to recover quickly once water is available again via the rehydration process [5–7].
Although signal reception and the transduction pathway of dehydration have been extensively reported, they remain largely unknown for the rehydration process. Using a DNA microarray, Oono et al.  found that rehydration-responsive genes included genes related to both stressed status release and growth recovery in Arabidopsis. In addition, ethylene-biosynthetic genes and genes responsive to jasmonic acid, gibberellin, and auxin were activated by rehydration, suggesting hormone balance is involved in the water recovery of plants.
In plants, the mitogen-activated protein kinase (MAPK) cascade plays an essential role in the signaling pathway of multiple abiotic and biotic stress cues [1, 2, 8–11]. The MAPK cascade is initiated by a mitogen-activated protein kinase kinase kinase (MAPKKK, MAP3K, or MEKK), which reversibly phosphorylates a mitogen-activated protein kinase kinase (MAPKK, MAP2K, or MKK) and subsequently phosphorylates the mitogen-activated protein kinase (MAPK or MPK). Then, MPK phosphorylates its downstream target proteins to modulate various developmental and physiological processes . In Arabidopsis, several components of the MAPK cascade, such as MPK4 and MPK6, were reported to be activated by drought [13, 14]; a recent report showed that MPK6 could be activated by drought, but inactivated by rehydration in Arabidopsis seedlings .
Cut roses (Rosa hybrida) are an important ornamental crop globally. Dehydration is a considerable postharvest problem for cut roses because their market supply is highly dependent on long-distance transportation, resulting in severe dehydration for this duration. Recently, we reported that in rose (R. hybrida) flowers, rehydration following dehydration triggered rapid, but transient, ethylene production in the gynoecia, namely all the carpels and pistils in a flower . During dehydration and rehydration, the protein level of a specific MAP kinase, RhMPK6, was maintained at a constant high level. However, RhMPK6 activity was only detected within 1 h of rehydration . Furthermore, activated RhMPK6 phosphorylated and stabilised RhACS1, a rate-limiting enzyme of ethylene biosynthesis, and resulted in an ethylene burst in gynoecia. Ethylene plays an important role in flower opening and senescence in roses [17–20]; thus, we speculated that the RhMPK6-RhACS1 module might be crucial to sense the rehydration signal and transmit it to ethylene to regulate flower opening and senescence in roses.
Previously, we found that a MAPKK protein, RhMKK9, could phosphorylate the RhMPK6 protein in vitro . However, to date it is unknown whether RhMKK9 is the actual upstream component activating RhMPK6 in gynoecia during rehydration. In the present study, we isolated the possible MAPK kinases upstream from RhMPK6 from roses and monitored their expression pattern during dehydration and rehydration. We found that RhMKK9 is the specific MAPK kinase that phosphorylates RhMPK6 and is responsible for the rehydration-induced ethylene production in gynoecia. Furthermore, methylation-sensitive PCR showed that DNA methylation of the promoter of RhMKK9 contributes to rehydration-induced upregulation of RhMKK9 expression.
RhMKK9 expression is rapidly and strongly induced by dehydration in rose gynoecia
We previously reported that ethylene production could be rapidly but transiently induced by rehydration in rose gynoecia when the flowers were subjected to dehydration-rehydration treatment . Moreover, temporal- and spatial-specific activation of an RhMPK6-RhACS1 cascade is responsible for this rehydration-induced ethylene production .
Silencing of RhMKK9 suppressed ethylene production and petal senescence in rose flowers after dehydration and rehydration treatment
In addition, we monitored the accumulation pattern of RhMPK6 transcript and protein, as well as the kinase activity of RhMPK6 in RhMKK9-silenced flowers. We tested the RhMPK6 activity and protein level after 30 min of rehydration and RhACS1 protein level after 1 h of rehydration, respectively. This was based on the results from our previous study, which showed that the highest level of RhMPK6 activity and RhACS1 protein appeared at 30 min and 1 h of rehydration, respectively . As expected, the transcript and protein level of RhMPK6 were not affected by RhMKK9 silencing. However, phos-tag SDS-PAGE demonstrated that RhMKK9 silencing largely weakened RhMPK6 phosphorylation during rehydration (Fig. 3b). Thus, we considered that low level of RhMPK6 activity caused low level of RhACS1 protein and consequentially low ethylene production in RhMKK9-silenced flowers during rehydration. Interestingly, we also detected a weak level of RhMPK6 kinase activity in RhMKK9-silenced lines. This could be attributed to the activity of residual RhMKK9 kinase because the RhMKK9-silenced lines were knock-down instead of knock-out lines. These results confirmed that RhMKK9 functioned upstream from RhMPK6 during rehydration.
Silencing of RhMKK9 downregulated the expression of genes associated with senescence and induced by ethylene in petals
DNA methylation status of the RhMKK9 promoter and gene body is altered during rehydration
Despite its simple structure, ethylene is functionally complex and plays a crucial role in a broad spectrum of plant developmental and environmental responses [33, 34]. Ethylene acts as an essential mediator in post-pollination fertilisation and ovule development, as well as associated petal senescence in various plants [35–39]. In Arabidopsis, ethylene was reported to be indispensable for fertilisation by inducing synergid cell death and establishing pollen tube block . In Phalaenopsis, a pollination-induced ethylene burst in the stigma and style ensured appropriate ovary development, and considerably promoted perianth senescence [35, 37]. Similarly, in carnation (Dianthus caryophyllus) flowers, removal of gynoecia repressed the production of ethylene and delayed petal senescence .
Among the various abiotic stresses, dehydration is known to influence ethylene production in plants, although the underlying regulatory mechanism seems complex. In plants such as cotton (Gossypium hirsutum L.) petioles and bolls [41, 42], wheat (Triticum aestivum L.) leaves , orange (Citrus sinensis Osbeck) flowers , as well as persimmon (Diospyros kaki Thunb.), and avocado (Persea americana Mill.) fruit, dehydration induces ethylene production [45, 46]. In detached persimmon fruit, water-loss induced ethylene burst in the calyx triggered ethylene production in other tissues and accelerated fruit ripening and softening .
In rose flowers, dehydration gradually increased ethylene production in sepals, whereas rehydration following a period of dehydration triggered rapid ethylene production in gynoecia . Moreover, the ethylene production in gynoecia, although very transient, is essential for the flower to fully open, suggesting that ethylene is naturally required for the water recovery of dehydrated flowers . Considering that continuous ethylene production is very harmful to flowers, the timing of ethylene production should be tightly regulated. Biochemical analysis demonstrated that protein phosphorylation-dependent accumulation of RhACS1, a rate-limiting enzyme of ethylene biosynthesis, is responsible for controlling the timing of ethylene production. In addition, RhACS1 protein phosphorylation is attributed to RhMPK6, a MAP kinase, which is also precisely regulated by rehydration in rose flowers . More important, RhMPK6 abundance is retained at a relatively constant level during both dehydration and rehydration, whereas RhMPK6 activation only occurs within 1 h of rehydration. Thus, these results indicate that appropriate ethylene production is important for the natural recovery process of rose flowers during rehydration. Therefore, it is not necessary for constant RhMPK6 accumulation during dehydration and rehydration, which is an energy-consuming process. Moreover, because MAPKs must be activated by MAPK KINASEs, it is reasonable to speculate that an upstream component should be involved in the rehydration-activation of RhMPK6.
In Arabidopsis, MPK6 has been reported to be activated by different MAPK kinases in response to various biotic and abiotic stresses [47, 48]. Cold and salinity stress caused activation of MPK6 is, at least partially, dependent on MKK2 [49, 50], whereas MKK5 is required for oxidative-triggered MPK6 [51, 52]. For drought stress, although MPK6 is activated by drought-induced ROS accumulation , the corresponding MAPK kinase has not been identified. Expression of the active version of MKK9 promotes MPK3 and MPK6 kinase activity and ethylene production, and enhances the sensitivity of transgenic Arabidopsis to salt stress .
Here, we isolated the homologues of MKK2, MKK4, MKK5, and MKK9 from roses and detected their expression during dehydration and rehydration. It is noteworthy that RhMKK9 expression, which could phosphorylate RhMPK6 in vitro , was relatively lower during dehydration, but significantly and sharply induced at the onset of rehydration, and then rapidly decreased to similar level before rehydration. Furthermore, specific silencing of RhMKK9 led to a significant reduction of rehydration-induced ethylene production in gynoecia. The rehydration-responsive expression pattern and function identification suggested that RhMKK9 is possibly the upstream component regulating the rehydration-activated RhMPK6-RhACS1 cascade and ethylene production in rose gynoecia. Interestingly, RhMKK9 silencing delayed both flower opening and senescence, further supporting that rehydration-caused ethylene production accelerated flower opening and senescence. Detection of gene expression showed that rehydration led to the rapid expression elevation of several ethylene-inducible genes, which were involved in abiotic stress-response and senescence, in TRV control flowers. Consistently, expression of ethylene-inducible genes was significantly inhibited in RhMKK9-silenced flowers. It is worth noting that the expression of two transcription factor genes, RhWRKY40 and RhMYB108, decreased after 12 h of rehydration in TRV controls to a relatively low level, similarly to RhMKK9-silenced flowers. Therefore, RhWRKY40 and RhMYB108 might be closely associated with ethylene production. However, expression of the senescence-associated RhSAG12 gene in the TRV control was significantly higher than that in RhMKK9-silenced flowers at 12 h after rehydration, implying that rehydration-caused ethylene production might also initiate the petal senescence process.
In the last decade, increasing evidence has shown epigenetic modification plays crucial roles in tolerance, adaption, and memory of plants to various abiotic stresses [27–30]. In Arabidopsis, histone modification of H3K4me3 and H3K9ac were enriched in drought stress-induced genes [6, 30, 55]. In the rehydration process, H3K9ac was rapidly removed, correlating to the inactivation of drought-inducible genes. Interestingly, H3K4me3 was removed more slowly than the H3K9ac mark, suggesting that H3K4me3 may be responsible for the epigenetic memory of drought [6, 30]. In rice, drought stress induced gene expression of the histone acetyltransferase (HATs) family and enhanced acetylation of H3K9, H3K18, H3K27, and H4K5 .
Dynamic DNA methylation and demethylation has also been broadly reported to be involved in plant responses to abiotic stresses [57–61]. In maize, cold stress-induced ZmMI1 gene expression is associated with DNA hypomethylation in the nucleosome cores . Here, we found that rehydration also resulted in hypomethylation on the RhMKK9 promoter, whereas elevated methylation on the RhMKK9 gene body. The factors involved in this rapid methylation and de-methylation process should be the subject of further studies.
In summary, a MAPK KINASE, RhMKK9, is the upstream component responsible for activating the RhMPK6-RhACS1 cascade. RhMKK9 expression was specifically and rapidly induced by rehydration in gynoecia. RhMKK9 silencing inhibited rehydration-caused ethylene bursts and delayed flower opening and senescence. In addition, we found that changes of DNA methylation status on the RhMKK9 promoter and gene body were associated with RhMKK9 induction by rehydration. These results explained how the flower, the reproductive organ, could quickly recover from dehydration using ethylene as a mediator.
Flowers of R. hybrida ‘Samantha’ were provided by a commercial greenhouse (Sunstone Company) in the Changping District, Beijing. The flowers were harvested at opening stage 2 as described previously . The flowers were immediately placed in tap water and transported to the laboratory within 1 h. Stems of the rose flowers were re-cut underwater to approximately 25 cm, and then were kept in deionised water (DW) until further processing.
Dehydration and rehydration treatments
Dehydration treatment was conducted as described previously [7, 16]. Flowers were placed horizontally on the bench in a climate-controlled room at 25 °C, 40–50% relative humidity, and a continuous light with intensity of 140 μmol m−2 s−1. The dehydration status was defined by fresh weight loss, and the flowers were subjected to rehydration when the flowers lost ~30% fresh weight. After the dehydration treatment, the bottom of flower stems were re-cut to remove about 1 cm under water to prevent air embolisms, and the flowers were placed in deionised water for rehydration. The flower phenotype was observed at different time points: 0, 1, 2, and 3 d after rehydration. After treatment, the flowers were cut open and the gynoecia, including carpels and pistils, were sampled as described previously [7, 16, 20]. Half of the gynoecia at D16 (dehydration for 16 h) and R1 (rehydration for 1 h) were taken for the ethylene production test, whereas the petals and the other half of the gynoecia were collected and frozen using liquid nitrogen and then were stored at −80 °C for RNA isolation.
Ethylene production measurement
The gynoecia were weighed and then placed in a 25 ml airtight GC vial, and the vials were kept for 30 min at 25 °C. From each vial, 2 μl headspace gas was withdrawn to measure ethylene concentration using a gas chromatograph equipped with a flame ionisation detector (GC-17A, Shimadzu, Japan) as described previously [7, 20]. After ethylene measurement, the gynoecia were dried in an oven at 60 °C to determine their dry weight, and then the ethylene production was calculated. Fifteen flowers were used for each time point.
Alignment of multiple deduced amino acid sequences was constructed using ClustalW2 software and visualised using the BioEdit program. Phylogenetic analysis was performed using MEGA 5.2.
RNA extraction, semi-quantitative RT-PCR analyses
Total RNA of petals was isolated using the hot borate method as described previously , and the total RNA of gynoecia was extracted using a RN38-EASY RNA extraction kit (Aidlab, Co, Ltd., Beijing, PRC). Total RNA was treated by RNase-free DNase I to avoid genomic DNA contamination. An volume of 1 μg of clean RNA was used to synthesise cDNA using M-MLV reverse transcriptase (Promega Corp., Madison, WI, USA) according to the manufacturer’s instructions. The rose ubiquitin gene (RhUBI, JK622648) was used as the internal control. The primers used in the RT-PCR analysis are listed in Additional file 1: Table S2. PCR reactions were carried out for 5 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 58 °C, 30 s at 72 °C, and followed by a supplemental incubation for 7 min at 72 °C for all the genes. The PCR products were separated on 1.5% agarose gels and visualised by ethidium bromide staining. Absolute values for transcript abundance from RT-PCR were quantified using the Alpha EaseFCTM 2200 software (Alpha Innotech, USA, Version 3.2.1). For all experiments, an individual flower was considered as a biological replicate and all experiments were performed with at least three replicates.
RhMKK9 gene silencing was performed as previously described . A 490-bp fragment from the RhMKK9-specific 3′ end (partial ORF and entire 3′UTR) was used to construct pTRV2-RhMKK9. The resulting constructs pTRV2-RhMKK9, pTRV1, and pTRV2 were transformed into Agrobacterium tumefaciens strain GV3101. Briefly, Agrobacterium was grown in LB broth containing 50 μg ml−1 kanamycin and 50 μg ml−1 gentamycin sulphate at 28 °C with shaking at 200 rpm overnight. These cultures were then diluted 1:50 v/v in fresh LB broth containing 10 mM MES, 20 mM acetosyringone, 50 μg ml−1 kanamycin, and 50 μg ml−1 gentamycin sulphate, and grown overnight as described above. Agrobacterium cells were harvested by centrifugation, and the pellet was suspended in infiltration buffer (10 mM MgCl2, 150 mM acetosyringone, and 10 mM MES, pH 5.6) to a final A600 of 1.8. A mixture of Agrobacterium cultures containing pTRV1 and pTRV2-RhMKK9 at a ratio of 1:1 (v/v) was used for rose transformation, and a mixture containing pTRV1 and pTRV2 was used as the negative control. The mixtures were stored at room temperature for 4 h in the dark prior to vacuum infiltration.
For vacuum infiltration, the flowers were placed upside down in a container (81.64 L), with the whole flower immersed into the prepared infiltration mixture. The flowers were then infiltrated by vacuum at 30 mmHg twice, each for 2 min. Then they were briefly washed with DW and kept in DW for 3 d at 8 °C before the dehydration treatment .
Immunoblot and kinase activity assays
Gynoecia protein was extracted as described previously . The following antibodies were used: goat polyclonal IgG anti-ACC synthase 6 antibody (SC-12771, Santa Cruz Biotechnology, http://www.scbt.com) to detect RhACS1; polyclonal anti-MPK6 (Sigma-Aldrich, http://www.sigmaaldrich.com) to detect RhMPK6. Secondary antibodies (horseradish peroxidase-conjugated goat anti-mouse IgG, Sigma-Aldrich) were used as recommended in the manufacturer’s instructions.
For the kinase activity assay, SDS-PAGE gel was supplied with 25 μM Phos-tag (Wako Chemicals) and 50 μM Zn+ was used as described previously [64–66]. The running buffer contained 100 mM Tris, 100 mM MOPS, and 0.1% (w/v) SDS. Sodium bisulphite was added to 5 mM immediately before electrophoresis.
DNA methylation assay
DNA methylation status was analysed by Chop-PCR as described previously . For Chop-PCR, genomic DNA (500 ng) was digested with the methylation-sensitive restriction enzyme Alu I, or by the methylated DNA-digesting enzyme McrBC for 3 h. The digested DNA was used as a template to amplify the RhMKK9 promoter and gene body. Undigested genomic DNA was amplified as an internal control.
1-aminocyclopropane-1-carboxylic acid synthase
Mitogen-activated protein kinase
Mitogen-activated protein kinase kinase kinase
Mitogen-activated protein kinase kinase
Open reading frame
Tobacco rattle virus
Virus-induced gene silencing
We thank Dr Pedro Nunes from Shanghai Center for Plant Stress Biology for his technical advice in Chop-PCR and excellent suggestions in writing.
This work was supported by the National Natural Science Foundation of China, (Grant 31130048 and 31372095), and the 948 project (2011-G17) of the Ministry of Agriculture of China.
Availability of data and materials
All data supporting the findings is contained within the manuscript and supplemental data sections. The complete coding sequences of RhMKK2, RhMKK4, RhMKK5, and RhMKK9 is available at NCBI under accession numbers KP269070, KP269071, KP269072, and KP269073, respectively.
NM and JG conceived and designed the study. JC and QZ performed phenotypic analysis, and VIGS, QW, and YM performed the gene expression analyses. MF, YL, and ZM performed immunoblot analyses of RhMPK6 and RhACS1, and the phosphorylation assay of RhMPK6. YZ and GL performed the experiments of Chop-PCR. HW was also involved in gene expression analysis. All authors have read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
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