Summary
Ethylene (ETH) controls climacteric fruit ripening and can be triggered by osmotic stress. However, the mechanism regulating ETH biosynthesis during fruit ripening and under osmotic stress is largely unknown in apple (Malus domestica).
Here, we explored the roles of SnRK2 protein kinases in ETH biosynthesis related to fruit ripening and osmoregulation. We identified the substrates of MdSnRK2‐I using phosphorylation analysis techniques. Finally, we identified the MdSnRK2‐I‐mediated signaling pathway for ETH biosynthesis related to fruit ripening and osmoregulation.
The activity of two MdSnRK2‐I members, MdSnRK2.4 and MdSnRK2.9, was significantly upregulated during ripening or following mannitol treatment. Overexpression of MdSnRK2‐I increased ETH biosynthesis under normal and osmotic conditions in apple fruit. MdSnRK2‐I phosphorylated the transcription factors MdHB1 and MdHB2 to enhance their protein stability and transcriptional activity on MdACO1. MdSnRK2‐I also interacted with MdACS1 and increased its protein stability through two phosphorylation sites. The increased MdACO1 expression and MdACS1 protein stability resulted in higher ETH production in apple fruit. In addition, heterologous expression of MdSnRK2‐I or manipulation of SlSnRK2‐I expression in tomato (Solanum lycopersicum) fruit altered fruit ripening and ETH biosynthesis.
We established that MdSnRK2‐I functions in fruit ripening and osmoregulation, and identified the MdSnRK2‐I‐mediated signaling pathway controlling ETH biosynthesis.
Keywords: ACO1, ACS1, apple, ethylene, HB transcription factor, osmotic stress, SnRK2‐I
Introduction
Ethylene (ETH) is an important hormone involved in the regulation of plant growth, fruit ripening, and stress responses (Gane, 1934; Chang, 2016; Park et al., 2021). Fruits are classified as climacteric and nonclimacteric based on whether or not ETH controls ripening (Adams‐Phillips et al., 2004). Tomato (Solanum lycopersicum) and apple (Malus domestica) are typical climacteric fruits; their ripening is controlled mainly by ETH (Giovannoni, 2004; Tatsuki, 2010; Gapper et al., 2013). Ethylene biosynthesis also is associated with stress (Dubois et al., 2018). However, the regulatory mechanisms of both ripening‐ and stress‐related ETH biosynthesis are largely unclear in climacteric fruits.
ETH biosynthesis is controlled by two types of enzymes: 1‐aminocyclopropane‐1‐carboxylic acid synthases (ACSs) and 1‐aminocyclopropane‐1‐carboxylic acid oxidases (ACOs) (Yang & Hoffman, 1984; Kende, 1993). ACSs and ACOs have been identified in many species, but it is unclear which ACO and ACS members are involved in certain biological events (Booker & DeLong, 2015; Houben & Poel, 2019). The specific ACO and ACS genes promoting ETH biosynthesis have been identified in tomato and apple fruits. SlACS2, SlACS4, SlACS1A, SlACO1 and SlACO4 are the major ETH biosynthesis genes in tomato fruits, whereas MdACS1, MdACS3A and MdACO1 are key ETH biosynthesis genes in apple fruits (Nakatsuka et al., 1998; Oraguzie et al., 2004; Wakasa et al., 2006; Cara & Giovannoni, 2008; Wang et al., 2009).
Post‐translational regulation of ACS proteins is a major pathway controlling ETH biosynthesis, and expression levels of ACO1 correspond well with ETH production (Pattyn et al., 2021). Phosphorylation mediated by mitogen‐activated protein kinase 3/6 (MAPK3/6), 14‐3‐3 proteins and target of rapamycin kinase (TOR) affect the turnover of ACS proteins, thereby regulating ETH biosynthesis in Arabidopsis thaliana (Joo et al., 2008; Han et al., 2010; Yoon & Kieber, 2013; Zhuo et al., 2020). A calcium‐dependent protein kinase (CDPK), SlCDPK2, is also involved in ETH biosynthesis; it phosphorylates SlACS2 in tomato fruits (Kamiyoshihara et al., 2010). However, the post‐translational regulatory mechanisms of ACS proteins in climacteric fruits remain unknown. Moreover, it is unknown whether other protein kinases are involved in ACS protein regulation. The transcriptional regulation of ACO1 has been well‐studied in apple and tomato fruits (Lin et al., 2008; Li et al., 2017). MdMYC2, MaERF11, MaMADS7, SlHB1 (LeHB‐1), SlRIN and SlNAC9 are important transcription factors that directly bind to the promoter of ACO1 to improve its expression, thereby increasing ETH biosynthesis to accelerate fruit ripening (Ito et al., 2008; Lin et al., 2008; Xiao et al., 2013; J. Liu et al., 2015; M. Liu et al., 2015; Kou et al., 2016; Li et al., 2017). However, it is unclear how these transcription factors respond to ripening to drive ACO1 expression and whether other signals or components participate in this process.
In addition to ripening, various stresses also trigger ETH production in plants (Skirycz et al., 2011; Zhang et al., 2016; Savada et al., 2017; Dubois et al., 2018). Among these stresses, drought, flooding and salt stress are closely related to osmotic stress (Skirycz et al., 2011; Dubois et al., 2018); however, it is unclear how osmotic stress triggers ETH biosynthesis (Verma et al., 2016; Dubois et al., 2018). Sucrose nonfermenting protein kinase 1‐related protein kinase 2 (SnRK2) members function in response to osmotic stress (Zhang et al., 2011; Fujii & Zhu, 2012; Fujita et al., 2013). SnRK2 is a plant‐specific family of protein kinases that has three subfamilies, SnRK2‐I, SnRK2‐II and SnRK2‐III (Hrabak et al., 2003; Fujii & Zhu, 2012). Previous studies of SnRK2 focused mainly on the regulatory mechanisms of SnRK2‐III members in abscisic acid (ABA)‐related biological events and osmotic responses (Zhang et al., 2011; Fujii & Zhu, 2012; Fujita et al., 2013). Recent studies showed that ABA‐unresponsive subfamily I SnRK2s (SnRK2‐I) also play important roles in osmotic stress responses by activating components of the mRNA decapping complex VARICOSE (VCS) and interacting with PA to phosphorylate two dehydrin proteins, EARLY RESPONSE TO DEHYDRATION (ERD)10 and ERD14 (Maszkowska et al., 2019). SnRK2 family members have been identified in apple and tomato (Sun et al., 2011; Chen et al., 2013), and SlSnRK2s have been proposed to regulate tomato fruit ripening (Sun et al., 2011). However, the specific roles of SnRK2 family members in both apple and tomato remain unclear. Whether and which SnRK2s are involved in fruit ripening and ETH biosynthesis, and whether SnRK2‐mediated osmotic responses are related to ETH biosynthesis require further study.
In this study, we established that MdSnRK2‐I and SlSnRK2‐I protein kinases are important regulators of fruit ripening, synergistically regulating ETH biosynthesis. During fruit ripening or under osmotic stress, the MdSnRK2‐I members MdSnRK2.4 and MdSnRK2.9 are activated and then phosphorylate the HB transcription factors MdHB1 and MdHB1 to increase MdACO1 expression, thus enhancing ETH biosynthesis. In summary, we reveal a pathway controlling ETH biosynthesis during fruit ripening and under osmotic stress in apple fruit and demonstrate that SnRK2‐I is the key post‐translational regulator of ETH in response to ripening and osmotic stress in apple and tomato fruits.
Materials and Methods
Plant materials and growth conditions
Apple (Malus domestica cv Golden Delicious) were chosen for this study because their genome has been sequenced (Supporting Information Fig. S1a). Apple calli were obtained and cultured according to reported protocols (Alayón‐Luaces et al., 2008; Li et al., 2016). Apple calli were subcultured at 10‐d intervals before being subjected to gene transformation. ‘Micro Tom’ tomato plants were cultured according to reported protocols (Sun et al., 2011).
Bioinformation analysis and cloning of Sucrose nonfermenting protein kinase 1‐related protein kinase 2 (SnRK2) and HB genes
Bioinformation analysis, including phylogenetic tree construction and protein sequence alignments, was conducted as described previously (Han et al., 2015). To identify specific members of the MdSnRK2 and MdHB gene families, the coding sequences of the AtSnRK2 and AtHB genes were used as queries in a Blast search against the apple genome (http://genomics.research.iasma.it/) and NCBI database (https://www.ncbi.nlm.nih.gov/), yielding a total of nine members of the MdSnRK2 family (designated MdSnRK2.1‐MdSnRK2.9) and MdHB family (designated MdHB1–MdHB23).
Screening of SnRK2 and HB genes involved in ETH production
The full‐length cDNAs of SnRK2s and HBs were cloned into a pCambia1304 overexpression vector and then individually transformed into Agrobacterium tumefaciens strain EHA105 (Lazo et al., 1991). The EHA105 strain was cultured as described previously (Han et al., 2015). The transformed calli were used to detect ETH production and gene expression.
The cloned genes were further infiltrated into tomato fruit by injection at c. 25 d after fruit set. The fruits were then monitored throughout development and ripening. Ten to 15 pairs of fruit were infected for each gene with the empty vector (EV) as a control. The quantitative reverse transcription (qRT)‐PCR primers are listed in Tables S3 and S4.
Measurement of ETH production
Apple calli, apple fruit and tomato fruit samples (2 g callus or 5–8 pairs of fruit) were collected from the medium or the plants at each sampling timepoint, and were enclosed in gas‐tight containers (50 ml or 2.5 l) equipped with a sampling valve. The ETH released was determined as described previously (Li et al., 2015).
Quantitative reverse transcription PCR
The qRT‐PCR was performed as described by Han et al. (2015). Three biological replicates were set up, and each sample (five fruit or 3 g callus combined as one sample) was analyzed at least in triplicate. The primers used are listed in Tables S3 and S4.
Analysis of MdSnRK2s expression in response to different treatments
At 105 d post‐anthesis (DPA), fruit disks (10 mm diameter, 1 mm thickness) were prepared from six fruit and combined to make one disk sample (5 g) per treatment. The disc samples were first vacuum‐infiltrated for 30 min in equilibration buffer (Archbold, 1999) consisting of 50 mM MES‐Tris (pH 5.5), 10 mM MgCl2, 10 mM EDTA, 5 mM CaCl2, 200 mM mannitol and 5 mM vitamin C. Then, samples were shaken for 6 h at 25°C in equilibration buffer containing either 6% mannitol, 100 µM ABA, 200 mM NaCl or 100 µM ACC (acetyl‐CoA carboxylase). After incubation, the samples were washed with double‐distilled water, frozen immediately in liquid N2, and kept at −80°C until used. Each individual analysis was conducted with three replicates.
Functional analysis of SnRK2s with stable transgenic tomato plants
The pCambia1304‐35S‐MdSnRK2.4/pCambia1304‐35S‐MdSnRK2.9 constructs were transformed into A. tumefaciens EHA105 for stable transformation of tomato following a protocol described previously (Sun et al., 2006; Kimura & Sinha, 2008). To examine the effect of the heterologous and overexpression of MdSnRK2.4/2.9 on plant growth and development, nine to 12 seedlings from four independent lines with relatively high expression of SnRK2 were examined. For gene expression analysis, two or three fruit from each individual plant were combined as an individual sample. The primers used are listed in Tables S3 and S4.
Virus‐induced gene silencing (VIGS) in tomato fruit
An 890‐bp fragment of the SlSnRK2.1 and SlSnRK2.2 genes (GenBank accession nos. AK329882 and AK327274, respectively) and a 568‐bp fragment of the ethylene‐insensitive protein 2 (SlEIN2) gene (GenBank accession no. AY566238) were PCR‐amplified from tomato cDNA sources and cloned into pTRV2 to generate pTRV2‐SlSnRK2.1, pTRV2‐SlSnRK2.2 and pTRV2‐SlIEIN2. For tomato fruit infiltration, the procedure described by Fu et al. (2005) was used.
Protein extraction
Total proteins were extracted from apple calli, apple fruit and tomato fruit using the following extraction buffer (pH 7.5): 100 mM Tris–HCl, 100 mM KCl, 10% glycerol, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1% Triton X‐100 and 1× protease inhibitor cocktail. Protein concentration was measured using a BCA Protein Assay Kit (ComWin Biotech, Beijing, China) and adjusted to the same level for each sample.
Antibody production
MdSnRK2.4‐ and MdSnRK2.9‐specific antibody against synthetic peptides (MKKIMGVHYKIPDH and PDYVHISQDCRHLL, respectively), and MdSnRK2 and SlSnRK2 common peptide (GRFSEDEARYFFQQ) were produced by Abmart (Shanghai, China). MdHB1/2 and LeHB1‐specific synthetic peptide (SEEDDGSDD) was produced by Beijing Huada. The specificity of the antibodies was tested using fruit total proteins.
Yeast‐two‐hybrid assays
Yeast two‐hybrid (Y2H) assays were performed using the Matchmaker GAL4‐based Two‐Hybrid System (Clontech, San Francisco, CA, USA) according to the manufacturer’s instructions. Constructs were produced by cloning MdSnRK2.4 or MdSnRK2.9 into the pGBKT7 vector and MdHB1 or MdHB2 into pGADT7. The primers used for the Y2H assays are provided in Tables S3 and S4.
Bimolecular fluorescence complementation assays
Bimolecular fluorescence complementation (BiFC) assays were performed using tobacco (Nicotiana tabacum) as described previously (Han et al., 2015). The coding sequences of MdSnRK2.4 and MdSnRK2.9 were amplified and cloned into the SPYNE‐YFPn vector, and those of MdHBs and MdEIN2 were cloned into SPYCE‐YFPc (YFP, yellow fluorescent protein). Fluorescence was examined 3 d post‐transformation using a confocal laser‐scanning microscope (Fluoview FV1000; Olympus, Tokyo, Japan). The primers used are listed in Tables S3 and S4.
Co‐immunoprecipitation assays
For co‐immunoprecipitation (Co‐IP) assays, pMDC83:MdHB1/2‐GFP and SPYNE:FLAG‐MdSnRK2.4/2.9 were co‐transformed into apple calli, with FLAG‐MdSnRK2.4/2.9 and pMDC83‐GFP being co‐transformed as a control (GFP, green fluorescent protein). Total proteins were extracted 3 d post‐transformation. An equal amount of anti‐FLAG antibody coupled protein A + G‐Sepharose beads was added to total protein samples and detected with anti‐GFP antibody.
Mass spectrometry assays
Total proteins extracted from apple calli were incubated with glutathione S‐transferase (GST)‐MdSnRK2.9 at 4°C for 4 h, and then the MdSnRK2.9 protein and its interacting proteins were immunoprecipitated and analyzed by LC‐MS/MS.
In order to identify putative phosphorylation sites of MdHB1 and MdHB2 by mass spectrometry, 5 µg MdHB1/2‐His and 10 µg GST‐SnRK2.4/2.9 purified protein were incubated in 30 µl protein kinase buffer (20 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 10 mM ATP) at 30°C for 30 min. The reaction mixtures were analyzed by LC‐MS/MS.
MdSnRK2 phosphorylation sites in apple flesh at different developmental stages were analyzed. Total proteins extracted from apple flesh at 85 and 105 DPA were immunoprecipitated with anti‐MdSnRK2.4/2.9 monoclonal antibody and then analyzed by LC‐MS/MS.
In vitro phosphorylation assays
ADP‐Glo Kinase Assays were performed according to the manufacturer's instructions (Promega, Fitchburg, WI, USA). Activity was assessed by measuring ATP with a luminometer (GloMax, Promega, Madison, WI, USA), and the average activity value ± SD is presented. Primers used are listed in Tables S3 and S4.
In‐gel kinase activity assays
In‐gel kinase activity assays were performed as described previously (Liu et al., 2017) on apple calli grown on control medium (MS agar with 3% sucrose) or on the same medium supplemented with 6% mannitol or 100 µM ACC. Radioactivity was quantified using a Typhoon 9410 imager (Molecular Dynamics, GE Healthcare, Pittsburgh, PA, USA).
Phos‐tag mobility shift assays
Phosphoprotein mobility‐shift assays with phos‐tag reagent (AAL‐107) were used to detect phosphorylated MdSnRK2.4/2.9, SlSnRK2, MdHB1/2 or LeHB1 protein as described previously (Liu et al., 2017). Apple calli or fruit were treated with or without 100 µM ACC or 6% mannitol for different durations, and total proteins were extracted. For the phosphorylation of MdHB1/2 or LeHB1, the extracted total proteins were immunoprecipitated with anti‐HB1 polyclonal antibody. For the analysis of MdSnRK2.4/2.9 phosphorylation level in apple flesh at different developmental stages, total proteins extracted from apple flesh were immunoprecipitated with anti‐MdSnRK2.4 or MdSnRK2.9 monoclonal antibody. Phosphorylated SlSnRK2 was analyzed using anti‐SnRK2 monoclonal antibody. These samples were incubated with or without calf‐intestinal alkaline phosphatase (CIP) at 37°C for 30 min, and then analyzed using 12% (w/v) SDS‐PAGE gel containing 50 μM phos‐tag and 100 μM MnCl2. Proteins were detected with specific antibodies.
Transcriptional activity assays
β‐glucuronidase (GUS) activity was measured using a fluorometric GUS enzymatic assay following a protocol published previously (Jefferson, 1987). The promoter sequence of MdACO1 (designated ProMdACO1) was cloned into pCambia1301 to generate the pCambia1301‐ProMdACO1:GUS reporter construct. The CDSs of MdSnRK2.4/2.9, MdHB1/2 and MdEIN2 were cloned to generate effector constructs. The constructs were transformed into apple calli grown at 27°C on Murashige & Skoog (MS) medium supplied with 6% mannitol or 100 µM ACC. Equal amounts of total proteins were used to evaluate the effects of different effectors on GUS activity. Each experiment was repeated three times. The primers are listed in Tables S3 and S4.
ChIP‐qPCR assay
Chromatin immunoprecipitation (ChIP) was performed on apple calli expressing MdHB1/2‐GFP or the pMDC83‐GFP EV as described previously (Gendrel et al., 2005). Total proteins were extracted from each sample (3 g) and the MdHB1/2 proteins were immunoprecipitated using anti‐GFP (ab290; Abclone, Boston, MA, USA) antibody. Antibodies (NoAbs) were omitted as a negative control.
The enriched DNA fragments were analyzed by qRT‐PCR using the primers listed in Tables S3 and S4. PCR reactions were performed in triplicate for each sample, and the expression levels were normalized to that of MdActin.
Electrophoretic mobility‐shift assay
Electrophoretic mobility‐shift assay (EMSA) was performed using a LightShift® Chemiluminescent EMSA Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Oligonucleotide probes of MdACO1 were synthesized and labelled with 5′‐biotin (Sangon Biotech, Shanghai, China). The probes used are listed in Tables S3 and S4.
Cell‐free protein degradation assay
A cell‐free protein degradation assay of MdHB1/2 and MdACS1 was performed as described previously (Liu et al., 2017). To detect the degradation of MdHB1/2, apple calli transfected with MdSnRK2.4/2.9‐OE were treated with 6% mannitol or 100 µM ACC, and total proteins were extracted. Equal amounts of total proteins were incubated with equal amounts of recombinant MdHB1/2‐His protein and 10 mM ATP for the indicated period. The proteins were separated by SDS‐PAGE and detected with anti‐His antibody. For MdACS1 protein degradation, 5 µg purified GST‐MdACS1 protein was incubated with 2.5 µg purified His‐MdSnRK2.4/2.9 and 10 mM ATP for the indicated periods. The proteins were separated by SDS‐PAGE and detected with anti‐GST antibody. The relative amounts of proteins were determined by densitometry and normalized to loadings determined by Actin using ImageJ (v.1.8.0).
Statistical analysis
Samples were analyzed in triplicate, and the data are presented as means ± SD. Data were analyzed using Student’s t‐tests or Tukey’s honestly significant difference (HSD) test implemented in Sas (v.8.1; SAS, Cary, NC, USA).
Results
MdSnRK2.4 and MdSnRK2.9 regulate ETH biosynthesis in apple fruit
In order to explore the regulatory mechanism of ETH biosynthesis in apple fruit, we first measured ETH content in developing ‘Golden Delicious’ apple fruit. Ethylene content in apple fruit followed a similar pattern to that in tomato fruit, another typical climacteric fruit (Fig. S1b; J. Liu et al., 2015; M. Liu et al., 2015). Apple fruit ETH content was relatively low during the early stages of development, started to increase after 60 DPA, increased rapidly after 105 DPA, peaked at around 130 DPA and then gradually decreased (Fig. S1a). This pattern indicates that 105 DPA is a key stage for ripening‐related ETH in ‘Golden Delicious’ fruit.
Transgenic apple fruit are hard to obtain and therefore fruit calli are used widely as a mock model to study the effects of target genes on fruit ripening‐related metabolic changes in apple fruit (Tatsuki, 2010; Dong et al., 2011). However, it is unclear whether ETH biosynthesis is induced in apple calli. Hence, we treated ‘Golden delicious’ calli with ETH and its biosynthesis precursor ACC at different concentrations to evaluate ETH biosynthesis in apple fruit cells (Adams & Yang, 1979). A low concentration (0.5 ppm) of exogenous ETH induced ETH biosynthesis in apple calli (Fig. S1c), indicating that apple callus cells are sensitive to ETH and can biosynthesize ETH effectively. ACC often is used to induce ETH responses (Adams & Yang, 1979; Guzmán & Ecker, 1990). We found that 5 and 100 µM ACC treatment significantly increased ETH biosynthesis in apple calli compared to a control treatment (NoACC), and the effects of ACC on NoACC production were similar to those of exogenous NoACC (Fig. S1c). Based on these results, we used ACC to induce the NoACC response in apple calli in subsequent experiments.
We identified nine MdSnRK2 genes that belong to three subfamilies in apple calli (Fig. S1d). In particular, MdSnRK2.1, MdSnRK2.4 and MdSnRK2.9 had high expression levels, especially at 105 DPA (Fig. 1a). We overexpressed all MdSnRK2s into apple calli and measured ETH biosynthesis. Overexpression of the subfamily I members MdSnRK2.4 and MdSnRK2.9 (MdSnRK2.4‐OE and MdSnRK2.9‐OE) resulted in the highest ETH levels (Fig. 1b). This indicates that MdSnRK2.4 and MdSnRK2.9 are involved in the regulation of ETH biosynthesis in apple fruit cells. Notably, although expression levels of MdSnRK2.4 and MdSnRK2.9 were consistently high during the ripening process, they did not increase with the ripening process (Fig. 1a). We then detected the protein and phosphorylation levels of MdSnRK2.4 and MdSnRK2.9 during apple fruit ripening. Although MdSnRK2.4 and MdSnRK2.9 protein levels decreased slightly with ripening (Fig. 1c), their phosphorylation levels were significantly upregulated from 85 DPA and then gradually increased throughout the ripening process (Fig. 1d). These results were consistent with an increase in activated phosphorylation sites of MdSnRK2.4 and MdSnRK2.9 from 85 to 130 DPA (Fig. S2; Table S1). Taken together, our results indicate that MdSnRK2‐I regulates ETH biosynthesis via phosphorylation during fruit ripening.
MdSnRK2.4 and MdSnRK2.9 regulate the expression of MdACO1 and degradation of MdACS1
1‐aminocyclopropane‐1‐carboxylic acid synthases (ACSs) and 1‐aminocyclopropane‐1‐carboxylic acid oxidases (ACOs) are the rate‐limiting enzymes in ethylene biosynthesis (Yang & Hoffman, 1984; Kende, 1993). To determine which genes are involved in MdSnRK2.4‐ and MdSnRK2.9‐meditated ETH biosynthesis, we analyzed the expression of MdACOs and MdACSs in MdSnRK2.4‐OE and MdSnRK2.9‐OE apple calli. The expression levels of MdACO1, MdACO2, MdACO3, MdACS1 and MdACS3A were significantly higher in MdSnRK2.4‐OE and MdSnRK2.9‐OE calli than in control calli. Moreover, the expression levels of MdACO1 were four‐ to six‐fold higher than those of other biosynthesis genes in MdSnRK2.4‐OE, MdSnRK2.9‐OE apple calli (Fig. 2a). MdACO1 is the major ACO gene controlling ethylene biosynthesis in apple fruit (Nakatsuka et al., 1998; Schaffer et al., 2007). These results indicate that MdACO1 is an important downstream biosynthesis gene affected by MdSnRK2.4 and MdSnRK2.9.
ACSs are involved in ETH biosynthesis via regulating their gene expression and phosphorylation‐mediated protein degradation (Park et al., 2021). We found that MdSnRK2.9 interacted with MdACS1 (Fig. 2b). The cell‐free degradation assay showed that co‐incubation of MdSnRK2.4‐His and MdSnRK2.9‐His could reduce the degradation of GST‐MdACS1. However, it did not obviously affect the degradation of GST‐MdACS12A, the phospho‐dead mutant of MdACS1 that was generated by inactivating SnRK2‐recognized phosphorylation sites (Fig. 2c).
Compared to ACSs, the regulatory mechanisms of ACOs, in particular their post‐transcriptional regulatory mechanism, remain largely unknown. Therefore, we focused on determining how MdSnRK2‐I induces the expression of MdACO1 to promote ETH biosynthesis.
MdSnRK2.4 and MdSnRK2.9 are activated by osmotic stress and ETH in apple fruits
As MdSnRK2.4 and MdSnRK2.9 are activated during fruit ripening (Fig. 1d), we next sought to determine whether these proteins are activated by ETH. We investigated the effects of ACC treatment on phosphorylation changes in MdSnRK2.4 and MdSnRK2.9 in apple flesh (Fig. 3a).
The SnRK2 family is reported to be widely involved in osmotic stress, which, in turn, induces ETH biosynthesis (Skirycz et al., 2011; Zhang et al., 2011; Fujii & Zhu, 2012; Dubois et al., 2018). Therefore, we investigated the effects of mannitol treatment on MdSnRK2.4 and MdSnRK2.9. A phos‐tag assay showed that the phosphorylation levels of MdSnRK2.4 and MdSnRK2.9 in apple flesh were higher under 6% mannitol and 100 µM ACC treatments than under the control treatment at 105 DPA (Figs 3a, S3a–c). Additionally, an in‐gel assay revealed that MdSnRK2.4 and MdSnRK2.9 were highly activated in MdSnRK2.4‐OE and MdSnRK2.9‐OE apple calli under 6% mannitol and 100 µM ACC treatments (Fig. 3b).
Then, we determined whether the mannitol‐ and ACC‐activated MdSnRK2.4 and MdSnRK2.9 could mediate ETH biosynthesis in apple calli. As shown in Fig. 3(c), ETH production in MdSnRK2.4‐OE and MdSnRK2.9‐OE apple calli was significantly higher under 6% mannitol and 100 µM ACC treatments compared with that under the control treatment. Moreover, co‐transformed MdSnRK2.4‐OE and MdSnRK2.9‐OE apple calli had higher ETH levels than single transformation calli (Fig. 3c). These results indicate that mannitol‐ and ACC‐activated MdSnRK2.4 and MdSnRK2.9 synergistically regulate ETH biosynthesis in apple fruit.
Notably, MdACO1 expression in MdSnRK2.4‐OE and MdSnRK2.9‐OE apple calli was more sensitive to 6% mannitol and 100 µM ACC than that in EV fruit calli, indicating that MdACO1 expression is the important output for MdSnRK2.4‐ and MdSnRK2.9‐mediated ETH biosynthesis under mannitol and ACC treatments (Fig. 3d).
MdSnRK2.4 and MdSnRK2.9 interact with and phosphorylate MdHB1 and MdHB2
In order to determine how MdSnRK2.4 and MdSnRK2.9 regulate MdACO1 expression, we investigated the substrates of MdSnRK2‐I by IP‐MS. More than 30 candidate proteins were identified, including one transcription factor (ID: V5LLY3) (Table S2). This transcription factor belongs to subfamily I of the HD‐Zip family and has a high sequence similarity with LeHB‐1, the reported regulator of ACO1 in tomato fruits (Fig. S4a,b; Lin et al., 2008). We named this transcription factor MdHB1. Then, we performed Y2H to examine how MdHB1 interacts with MdSnRK2.4 and MdSnRK2.9 (Fig. 4a). MdSnRK2.4 and MdSnRK2.9 interacted with the HOX (Homeobox) domain of MdHB1 via the kinase activity domain (Fig. 4a). Therefore, we identified 23 HD‐Zip I members in apple and determined their expression patterns in apple fruit during ripening. Of the HD‐Zip I members detected, MdHB1 and MdHB2 had the highest expression levels (Fig. S4c,d). Additionally, more ETH was produced in MdHB1‐OE and MdHB2‐OE apple calli than in EV apple calli (Fig. S4e). A BiFC assay showed that MdSnRK2.4 and MdSnRK2.9 interacted only with MdHB1 and MdHB2, and not with the other five HD‐Zip I proteins detected (Figs 4b, S4e,f). We also validated these interactions using Co‐IP by expressing MdHB1/2‐GFP and FLAG‐MdSnRK2.4/2.9 in apple calli (Fig. 4c). Taken together, these results indicate that MdSnRK2.4 and MdSnRK2.9 interact with MdHB1 and MdHB2 at their kinase activity domain in the nuclei of apple fruit (Fig. 4a–c, S4a–g).
The phos‐tag assay showed that the phosphorylation levels of MdHB1 and MdHB2 in apple calli also were increased by 6% mannitol and 100 µM ACC treatments (Fig. 4d). The expression level of MdACO1 in MdHB1‐OE and MdHB2‐OE apple calli was higher than those of other ETH biosynthesis genes, which is consistent with the MdACO1 expression pattern in MdSnRK2.4‐OE and MdSnRK2.9‐OE apple calli (Figs 2a, 4e). These results suggest that MdHB1 and MdHB2 are involved in mannitol‐ and ACC‐induced ethylene biosynthesis as the substrates of MdSnRK2.4 and MdSnRK2.9 (Figs 4d, S4b,h,i). We then examined whether MdSnRK2.4 and MdSnRK2.9 can phosphorylate MdHB1 and MdHB2 in vitro (Fig. 4f,g). MdSnRK2.4‐eGFP and MdSnRK2.4‐eGFP immunocomplexes, which were purified from MdSnRK2.4‐OE and MdSnRK2.9‐OE single‐ or co‐transformed apple calli, phosphorylated MdHB1‐His, MdHB2‐His and their mixed proteins in vitro (Fig. 4f,g). The 6% mannitol and 100 µM ACC treatments enhanced MdSnRK2.4‐ and MdSnRK2.9‐mediated phosphorylation levels of MdHB1‐His, MdHB2‐His and their mixed proteins (Fig. 4g). These results indicate that MdHB1 and MdHB2 act as substrates in a synergistic response to mannitol‐ and ACC‐activated MdSnRK2.4 and MdSnRK2.9.
We further examined the phosphorylation sites of MdMdHB1‐His and MdHB2‐His induced by GST‐MdSnRK2.9 using MS technology and found that MdSnRK2.9 phosphorylated MdHB1 at sites T81, T94 and T130, and MdHB2 at sites T83, S84, T96 and T132 (Fig. S4j,k). Then, we mutated all of the threonine and serine in these sites to alanine to generate the phospho‐dead proteins MdHB1T81A,T94A,T130A (MdHB13A) and MdHB2T83A,S84A,T96A,T132A (MdHB24A). Under the control treatment, the phosphorylation levels of MdHB13A‐His and MdHB24A‐His were increased slightly by MdSnRK2.4‐eGFP and MdSnRK2.9‐eGFP either alone or in combination (Fig. 4f,g). The 6% mannitol and 100 µM ACC treatments also increased the phosphorylation levels of MdHB13A‐His and MdHB24A‐His, but to a lesser extent than those observed for MdHB1‐His and MdHB2‐His (Fig. 4f,g). These results indicate that T81, T94 and T130 were the corresponding phosphorylation sites of MdHB1, and that T83, S84, T96 and T132 were those of MdHB2 for mannitol‐ and ACC‐activated MdSnRK2‐I. However, MdHB13A‐His and MdHB24A‐His were not completely inactivated, suggesting that MdHB1 and MdHB2 have other phosphorylation sites involved in MdSnRK2‐I‐mediated osmotic stress and ETH responses (Fig. 4f,g).
MdHB1 and MdHB2 are the downstream substrates of MdSnRK2.4 and MdSnRK2.9 and regulate MdACO1 expression to mediate ETH biosynthesis
In order to determine the effects of phosphorylation on the biological functions of MdHB1 and MdHB2, we overexpressed MdHB1‐eGFP, MdHB2‐eGFP, MdHB13A ‐eGFP and MdHB24A ‐eGFP in apple calli and measured ETH biosynthesis. MdHB1‐OE and MdHB2‐OE apple calli had significantly higher ETH biosynthesis levels than EV calli, particularly under 6% mannitol and 100 µM ACC treatments. However, ETH production in MdHB13A ‐OE and MdHB24A ‐OE apple calli was not significantly different from that in EV calli, except for that in MdHB24A ‐OE calli under the control treatment and MdHB34A ‐OE calli under the mannitol treatment (Fig. 5a).
In general, phosphorylation can regulate the biological function of transcription factors by altering their stability or transcriptional activity (Liu et al., 2017; Srivastava et al., 2020). Therefore, we determine whether phosphorylation could change the stability of MdHB1 and MdHB2 proteins. Incubation with protein extractions from MdSnRK2.4‐OE and MdSnRK2.9‐OE apple calli reduced the degradation rate of MdHB1 and MdHB2 mixed proteins (Fig. 5b). This was enhanced under mannitol and ACC treatments. Then, we determined the effects of phosphorylation on the transcriptional activity of MdHB1 and MdHB2 using ChIP‐PCR. MdHB1 and MdHB2 interacted with the promoter of MdACO1 by binding several elements. Among these elements, E1 (recognition element of transcription), E4 (TC‐rich defense and stress‐responsive element) and E6 (ETH‐responsive element) were used to further explore the interaction with MdHB13A and MdHB24A (Fig. 5c). ACC and mannitol treatments enhanced the interactions of MdHB1 and MdHB2, but not those of MdHB13A and MdHB24A with E1, E4 and E6 (Fig. 5d). Moreover, an EMSA showed that MdHB1 and MdHB2 directly bound E1, E4 and E6 in vitro and that the binding with MdHB13A and MdHB24A was weaker (Fig. 5e). These results indicate that MdSnRK2.4‐ and MdSnRK2.9‐mediated phosphorylation is important for the protein stability and transcriptional activity of MdHB1 and MdHB2 (Fig. 5b–e).
Next, we investigated the signaling transduction pathway of MdSnRK2.4‐ and MdSnRK2.9‐mediated ETH biosynthesis. We overexpressed MdSnRK2.4, MdSnRK2.9, MdHB1, MdHB2, MdHB13A and MdHB24A to determine their effects on MdACO1 expression in apple calli. MdACO1 expression was significantly higher in MdSnRK2.4‐OE, MdSnRK2.9‐OE, MdHB1‐OE, MdHB2‐OE and their kinase‐substrate co‐transformed combinations than in EV calli (Fig. 5f,g). However, under mannitol and ACC treatments, the MdACO1 expression in MdHB13A MdHB24A calli, either with or without MdSnRK2.4‐OE and MdSnRK2.9‐OE, was either similar, or only slightly higher, than that in EV calli (Fig. 5f,g). These results indicate that osmotic stress‐ and ETH‐activated MdSnRK2.4 and MdSnRK2.9 can phosphorylate MdHB1 at sites T81, T94 and T130, and MdHB2 at sites T83, S84, T96 and T132. This phosphorylation enhanced their protein stability and transcriptional activity to regulate MdACO1 expression and therefore control ETH biosynthesis in apple fruit (Figs 1, 2, 3, 4, 5).
MdSnRK2‐I and SlSnRK2‐I regulate tomato fruit ripening by mediating ETH biosynthesis
In order to further explore the roles of MdSnRK2‐I in climacteric fruit ripening, four independent MdSnRK2.4‐HE (heterologous expression) and MdSnRK2.9‐HE transgenic tomato lines were generated (Fig. S5a). Compared to EV fruit, ripening was significantly accelerated and ethylene biosynthesis was significantly increased in MdSnRK2.4‐HE and MdSnRK2.9‐HE fruit (Figs 6a,b, S5b). Similar to the effect in apple fruit, a 100 µM ACC treatment increased ETH biosynthesis in MdSnRK2.4‐HE and MdSnRK2.9‐HE tomato fruit to a greater extent than in EV fruit (Fig. 6c).
We then determined the expression levels of HB/ACO module genes in MdSnRK2.4‐HE and MdSnRK2.9‐HE tomato fruit. SlACO1, SlACO4 and SlHB1(LeHB‐1) expression levels were significantly higher in MdSnRK2.4‐HE and MdSnRK2.9‐HE tomato fruit than in EV fruit (Fig. 6d). These three genes are important biosynthesis genes controlling ETH‐mediated tomato fruit ripening (Nakatsuka et al., 1998; Cara & Giovannoni, 2008; J. Liu et al., 2015; M. Liu et al., 2015). In addition, compared with EV plants, MdSnRK2.4‐HE and MdSnRK2.9‐HE tomato plants were dwarfed, had twisted leaves and had shorter internodes with more branches (Fig. S5c,d). This is a typical phenotype associated with high levels of ETH (Li et al., 2021; Pattyn et al., 2021). Additionally, the leaves of transgenic plants turned yellow earlier than those of EV plants, indicating that MdSnRK2.4 and MdSnRK2.9 heterologous expression accelerated tomato plant senescence (Fig. 6a). Because stable transgenic expression affected plant growth, we transiently expressed MdSnRK2.4 and MdSnRK2.9 in tomato fruit to isolate the effect of MdSnRK2‐I on fruit ripening. The ETH content was significantly higher in MdSnRK2.4‐HE and MdSnRK2.9‐HE fruit than in EV fruit, and ripening was faster (Fig. 6e,f). These results suggest that MdSnRK2.4‐HE and MdSnRK2.9‐HE regulate tomato fruit ripening by affecting HB/ACO module‐mediated ETH biosynthesis.
Based on our findings, we hypothesize that SnRK2‐I and the HB/ACO module may conservatively regulate ethylene biosynthesis in both apple and tomato fruits (Figs 1, 2, 3, 4, 5, 6). Eight SnRK2 members have been identified in tomato, of which SlSnRK2.1 and SlSnRK2.2 are members of subfamily I (Fig. S1d; Sun et al., 2011). To determine the function of SlSnRK2.1 and SlSnRK2.2, we upregulated the expression of SlSnRK2.1 and SlSnRK2.2 using transient overexpression technology in tomato fruit. SlSnRK2.1‐OE and SlSnRK2.2‐OE fruit ripened faster and had significantly higher ETH production than EV fruit, whereas SlSnRK2.1‐VIGS and SlSnRK2.2‐VIGS fruit ripened slower and produced significantly less ETH than EV fruit (Fig. 7a,b). These results indicate that the SlSnRK2‐I subfamily is involved in regulating ETH biosynthesis and tomato fruit ripening. MdHB1, MdHB2 and their homolog SlHB1 (LeHB‐1) all belong to the HD‐Zip I subfamily (Fig. S4a). We blasted two conserved peptides of MdHB1 and MdHB2 bound with MdSnRK2‐I, and three conserved phosphorylation sites of MdHB1 and MdHB2 mediated by MdSnRK2‐I against the tomato genome. These conserved peptides and phosphorylation sites were carried only by LeHB‐1, and not by other HD‐Zip I subfamily members (Fig. S4b). We also found that SlSnRK2.1 and SlSnRK2.2, which were activated by mannitol and ACC treatments, could phosphorylate MdHB1 and MdHB2 in vitro (Fig. 7c). This suggests that the phosphorylation regulatory mechanisms of SlSnRK2‐I and MdSnRK2‐I in response to mannitol and ACC treatments might be specific for particular substrate structures.
Ethylene‐insensitive protein 2 is an important component controlling endogenous ETH signaling and thus regulating fruit ripening (Fu et al., 2005; Gao et al., 2016). To determine whether endogenous ETH affects the function of the SnRK2‐HB module, we determined phosphorylation levels of the SnRK2 family and SlHB1 (LeHB‐1) in SlEIN2‐VIGS tomato fruit. Phosphorylation levels of SnRK2 and SlHB1 (LeHB‐1) were reduced in SlEIN2‐VIGS fruit compared with in EV fruit, both with and without ACC treatment (Figs 7d,e, S5d). These results suggested that the SlSnRK2‐SlHB1 module acts downstream of SlEIN2 in tomato fruit.
Discussion
ETH is an important plant hormone with various biological functions. The regulatory mechanisms of ETH signal transduction and biosynthesis have been well‐established in Arabidopsis thaliana and crops such as maize (Zea mays) and rice (Oryza sativa) (Yang et al., 2015; Park et al., 2021). However, despite being the major hormone regulating the ripening of climacteric fruits, the regulation of its signal transduction and biosynthesis in fruit is largely unclear. In this study, we revealed a pathway mediated by phosphorylation that regulates ETH biosynthesis in apple fruit (Fig. 8). As shown in Fig. 8, the ABA‐unresponsive subfamily I of sucrose nonfermenting protein kinase 1‐related protein kinase 2 (MdSnRK2‐I) members MdSnRK2.4 and MdSnRK2.9 were activated during fruit ripening and under osmotic stress. Then, they phosphorylated specific sites of MdHB1 and MdHB2 transcription factors to increase their stability and transcriptional activity, thereby enhancing the expression of their downstream 1‐aminocyclopropane‐1‐carboxylic acid oxidase gene (MdACO1), which encodes the rate‐limiting enzyme of ETH biosynthesis in apple fruit. In addition to MdACO1, MdSnRK2.4 and MdSnRK2.9 interacted with another rate‐limiting enzyme, 1‐aminocyclopropane‐1‐carboxylic acid synthase 1 (MdACS1), enhancing its stability by phosphorylating two sites. The higher MdACO1 expression and enhanced MdACS1 protein stability increased ETH production, promoting ripening and mediating the plant's response to osmotic stress (Fig. 8).
SnRK2‐I, SnRK2‐II and SnRK2‐III modulate plant growth and abiotic stress responses, in particular, responses to drought‐ and salt‐triggered osmotic stress (Zhang et al., 2011; Fujii & Zhu, 2012; Fujita et al., 2013; Lin et al., 2020). Compared to SnRK2‐II and SnRK2‐III, little is known about the function, substrates and regulatory mechanisms of SnRK2‐I in higher plants (Maszkowska et al., 2021). SnRK2‐I is involved in responses to salt and osmotic stress by phosphorylating VARICOSE (VCS) and EARLY RESPONSE TO DEHYDRATION (ERD); however, its response to ABA was unknown (Fujii et al., 2011; Krzywińska et al., 2016; Lin et al., 2020). Additionally, it was unclear whether and how SnRK2‐I, SnRK2‐II, and SnRK2‐III are related to other plant hormones that function in osmoregulation (Maszkowska et al., 2021). Osmotic stress promotes ETH biosynthesis, and ACOS are important genes in this response (Skirycz et al., 2011; Zhang et al., 2016; Dubois et al., 2017, 2018; Savada et al., 2017). Our results indicate that osmotic stress activates MdSnRK2‐I to increase MdACO1‐mediated ETH biosynthesis in apple fruit (Fig. 3). These findings extend our understanding of the interactions between SnRK2s and plant hormones, as well as osmoregulation in higher plants. We also found that SlSnRK2‐I regulates ETH biosynthesis and is stimulated by osmotic stress in tomato fruit (Fig. 7). This observation suggests that SnRK2‐I may be conservatively involved in osmotic stress‐triggered ETH biosynthesis in climacteric fruits. However, whether this mechanism is conserved in other plants remains unclear. Additionally, SnRK2s are reported to be functionally redundant (Fujii et al., 2011; Lin et al., 2020). However, we established that MdSnRK2‐I and SlSnRK2‐I regulate ETH biosynthesis synergistically rather than redundantly. Whether SnRK2‐II and SnRK2‐III are involved in regulating ETH biosynthesis requires further study.
ETH controls the ripening of climacteric fruits (Chang, 2016; Park et al., 2021). Tomato fruit are a common model in ETH‐mediated ripening studies (Li et al., 2021; Park et al., 2021). Recently, our understanding of the transcriptional regulation of ETH‐mediated fruit ripening has improved (Barry et al., 2000; Alexander & Grierson, 2002; Cara et al., 2008; Gapper et al., 2013; Kou et al., 2016; Li et al., 2017). SlHB1 (LeHB‐1) is an important regulator of tomato fruit ripening and ACO1‐mediated ETH biosynthesis (Lin et al., 2008). MdHB1, which is homologous to SlHB1 (LeHB‐1), is a regulator of anthocyanin accumulation in apple fruit (Jiang et al., 2017). Our results showed that SlHB1 (LeHB‐1) has two homologs in apple, MdHB1 and MdHB2 (Fig. S4a,b). These transcription factors increased MdACO1 expression and therefore regulated ETH biosynthesis (Figs 5, S4e). Further analysis showed that MdHB1 and MdHB2 could be phosphorylated by MdSnRK2‐I, which enhanced their effects on MdACO1 by improving their protein stability and transcriptional activity (Figs 4, 5). These results reveal the post‐transcriptional regulatory mechanism of HB transcription factors in ETH‐mediated fruit ripening. Additionally, MdSnRK2‐I was found to regulate MdACS1 degradation (Fig. 2b,c), suggesting that MdSnRK2‐I can act as a molecular switch of these two rate‐limiting steps to control ETH biosynthesis efficiently. However, whether SnRK2‐I protein kinases fully control ETH biosynthesis will need to be verified in fruit whose genomes have been edited to have altered MdSnRK2‐I.
We also established that SlSnRK2‐I regulates tomato fruit ripening by affecting ETH biosynthesis (Fig. 7a,b). Fruit ripening is a complex process including the accumulation of pigment, sugar, acid, aroma compounds and other substances (Adams‐Phillips et al., 2004; Gapper et al., 2013; Li et al., 2021). Both ETH and osmotic stress affect fruit quality (Alexander & Grierson, 2002; Cara & Giovannoni, 2008; Li et al., 2021). Drought and salinity significantly reduce the yield and quality of apple fruit (Dong et al., 2011; Ma et al., 2017a,b). However, moderate drought can improve apple fruit quality (Ma et al., 2017a,b). Future studies should focus on identifying the SnRK2‐I substrates associated with fruit quality and clarifying the underlying role of SnRK2‐I in osmoregulation and ripening. This information could help to identify candidate genes/phosphorylation sites and thereby improve our understanding of the balance between stress responses and fruit quality (Zhang et al., 2011; Lin et al., 2020; Maszkowska et al., 2021). FaSnRK2.6, a member of the SnRK2‐III family, is a negative regulator of strawberry fruit ripening and anthocyanin accumulation (Han et al., 2015). Therefore, the role of other SnRK2s in ripening of climacteric and nonclimacteric fruits should be explored to fully understand post‐transcriptional regulation of ripening.
Author contributions
BL and WJ designed the study and conceived the experiments; WJ and SZ drafted the manuscript; MJ performed the experiments and analyzed the data; XL, WW, TL, ZD, YC, KZ, HZ, WM, QF, LL and JY assisted with some of the experiments; and BL and MJ wrote the manuscript.
Supporting information
Acknowledgements
We thank Prof. Don Grierson from the University of Nottingham and Prof. James J. Giovannoni from Cornell University for their critical comments and suggestions on the manuscript. We thank Prof. Daqi Fu from China Agriculutrual University for helping create ein2‐related materials. This work was supported by the National Key Research and Development Program (2018YFD1000201 and 2018YFD1000200), the National Natural Science Foundation of China (31772284, 32072551, 31672133 and 31872086), the 111 Project (B17043), the 2115 Talent Development Program of China Agricultural University, and the Construction of Beijing Science and Technology Innovation and Service Capacity in Top Subjects (CEFF‐PXM2019‐014207‐000032).
Contributor Information
Bingbing Li, Email: libingbing@cau.edu.cn.
Wensuo Jia, Email: jiaws@cau.edu.cn.
Data availability
The data that support the findings of this study are available on request from the corresponding author.
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Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author.