MdSnRK1.1 interacts with MdGLK1 to regulate abscisic acid-mediated chlorophyll accumulation in apple

Abstract Abscisic acid (ABA), as a plant hormone, plays a positive role in leaf chlorosis; however, the underlying molecular mechanism is less known. Our findings provide ABA treatment reduced the chlorophyll accumulation in apple, and Malus × domestica Sucrose Non-fermenting 1-Related Protein Kinase 1.1 (MdSnRK1.1) participates in the process. MdSnRK1.1 interacts with MdGLK1, a GOLDEN2-like transcription factor that orchestrates development of the chloroplast. Furthermore, MdSnRK1.1 affects MdGLK1 protein stability through phosphorylation. We found that Ser468 of MdGLK1 is target site of MdSnRK1.1 phosphorylation. MdSnRK1.1-mediated phosphorylation was critical for MdGLK1 binding to the target gene MdHEMA1 promoters. Collectively, our results demonstrate that ABA activates MdSnRK1.1 to degrade MdGLK1 and inhibit the accumulation of chlorophyll. These findings extend our understanding on how MdSnRK1.1 balances normal growth and hormone response.


Introduction
Due to the existence of abiotic stress, the survival of plants has been posed to a serious threaten [1].As a stress signal, abscisic acid (ABA) plays an important role in the defense against abiotic stress [2].Further study revealed that the ABA signaling regulatory network is very complicated, and the main ABA signaling pathway is comprised of a series of modification proteins, such as pyrabactin resistance/pyr1-like/regulatory components of ABA receptor (PYR/PYL/RCARs), type 2C protein phosphatases (PP2Cs), and SNF1-related protein kinase 2 s (SnRK2s).ABA binds to PYR and promotes interactions between the ABA-bound receptors and PP2Cs [3,4].With the decrease of PP2C activity, the inhibition of SnRK2 by PP2Cs also decreases, thereby reducing or eliminating the effect of PP2Cs on SnRK2.Subsequently, the freed SnRK2 phosphorylates downstream factors to enhance the expression of stress-or ABA-induced genes [5][6][7].As the orthologous gene of SnRK2, SnRK1 performs similar functions in the regulation of the ABA signaling pathway.PP2Cs interact with the SnRK1 catalytic subunit when ABA content is deficient.These PP2Cs dephosphorylate and inactivate SnRK1, thus participating in the ABA hormone signal pathway [8,9].
SnRK1 is an evolutionarily conserved heterotrimeric protein kinase complex including a catalytic α-subunit (SnRK1.1/1.2/1.3 in Arabidopsis) and regulatory β-and γ -subunits [10][11][12].SnRK1 functions as a Ser/Thr protein kinase that phosphorylates downstream proteins through a highly conserved T-loop residue (T175 in SnRK1.1)[13].Phosphorylation of SnRK1 is crucial to activate downstream transcription factors.As an energy effect factor, SnRK1 is often associated with coordinating various stressors [14].The kinase activity of SnRK1 is activated at low energy levels such as darkness, hunger, low nutrition, and osmotic stress [15,16].SnRK1 triggers massive transcriptional and metabolic processes by phosphorylating metabolic signaling molecules or enzymes to respond to low energy levels [17].Previous reports have shown that SnRK1 is associated with the ABA pathway through stress and metabolic signals [14].Evidence shows that SnRK1 has directly participated in ABA signaling in Arabidopsis [18].In tomato seeds, ABA differentially regulates the expression of SNF1-related kinase complex genes [19].The repressed SnRK1 gene leads to pea embryo maturation defects, which is a phenotype similar to abscisic acidinsensitive [20].The synthesized peptides of conserved motifs of ABA-responsive basic leucine zippers (bZIPs) [21], the recombinant abscisic acid-insensitive 5 (ABI5) and bZIP12 (also known as EEL or DPBF4) proteins can be phosphorylated by SnRK1 in Arabidopsis [22].bZIP63 is a downstream substrate of the SnRK1 catalytic subunit in Arabidopsis [23].Previous studies showed that MdSnRK1.1 transgenic apple materials are extremely sensitive to ABA treatment [24].Once ABA activates MdSnRK1.1,MdSnRK1.1 phosphorylates and affects the protein stability of MdCAIP1 [24].
In the growth and development process of Arabidopsis, ABA treatment induces leaf yellowing, reduces chlorophyll levels, and impairs chloroplast development [25,26].In Arabidopsis, the elevation of ABA levels leads to the low expression of chlorophyll biosynthesis genes, such as Glu tRNA reductase (HEMA1) and protochlorophyllide oxidoreductase (POR) [27].Moreover, the activity of constitutive photomorphogenic 1 (COP1) decreases with the suppression of chlorophyll content after long periods of ABA treatment [28].Specific protein interaction between COP1 and GLK1 has been found to play pivotal roles during the ABA treatment process [28].
GLK is a member of the v-myb avian myeloblastosis viral oncogene homolog (MYB) transcription factor family, which protein contains a C terminal GCT-box and a highly conserved DNA binding domain (DBD) [29].GLK transcription factors are involved in the regulation of chlorophyll accumulation and the synthesis of nuclear chloroplast-localized proteins in plants [29][30][31].In Arabidopsis, the size of the chloroplast and the numbers of thylakoid lamellae in glk1glk2 double mutants are both smaller than normal growth strain [14].In tomato, the high expression of SlGLK2 affects the chloroplast development and photosynthetic efficiency [32,33].
In this study we demonstrated that ABA treatment inhibited the accumulation of chlorophyll in apple leaves.We

ABA inhibits the accumulation of chlorophyll in apple leaves
ABA is confirmed to be involved in various abiotic stress processes in plants [2].Long-term application of ABA leads to chlorosis of plant leaves [25,26].To observe the phenotype of apple leaves under ABA treatment, apple leaves were treated with 0, 25, 50, 100, 150, 200, and 300 μM ABA for 0 to 30 days, and dimethyl sulfoxide (DMSO) was used as a control.We found that severe damage occurred to apple leaves after ABA treatment at the concentration of 200 and 300 μM.The most significant phenomenon of leaf chlorosis occurred when ABA concentration was 150 μM, and the total chlorophyll content was also the lowest (Fig. S1, see online supplementary material).The chlorophyll content and leaf color did not change after 15 days of ABA treatment compared to 30 days of ABA treatment.Therefore, 150 μM ABA concentration treatment for 15 days was used for subsequent apple leaf treatment.Chlorosis of apple leaves began to appear as ABA treatment time was extended (Fig. 1a).The chlorophyll content of leaves also gradually decreased as ABA treatment time was extended compared to the DMSO treatment group (Fig. 1b).The transcript level of the chlorophyll biosynthesis genes MdHEMA1, MdCAO, and MdCHLH also decreased gradually (Fig. 1c-e).The 15-day ABA-treated yellow plants turned green when transferred to DMSO plates (Fig. S2a, see online supplementary material).Correspondingly, the chlorophyll content recovered to the level of continuous DMSO treatment for 30 days (Fig. S2b, see online supplementary material).Therefore, the yellowing phenotype of apple leaves induced by ABA was caused by insufficient chlorophyll accumulation, which affected chloroplast development.

MdSnRK1.1 transcription is induced by ABA
SnRK1 participates in ABA signaling network in Arabidopsis [9].To clarify whether MdSnRK1.1 directly involves in ABA signaling pathway, the transcription level of MdSnRK1.1 was examined in ABA-treated apple plants.The expression of MdSnRK1.1 was detected when the GL-3 samples were treated with ABA at 0, 1, 3, 6, 12, and 24 h.The transcription level of MdSnRK1.1 was induced under ABA treatment, and induction was most obvious at 12 h (Fig. 2a).pMdSnRK1.1::GUStransgenic Arabidopsis plants were obtained to further detect the impact of ABA on the transcription level of MdSnRK1.1 (Fig. S3a, see online supplementary material).The GUS staining of ABA-treated pMdSnRK1.1::GUSseedlings deepened as the ABA concentration was increased (Fig. 2b).The GUS activity of the ABA-treated pMdSnRK1.1::GUSseedlings was higher than DMSO-treated seedlings (Fig. 2c).These results indicated that ABA induced the transcription level of MdSnRK1.1.

MdSnRK1.1 inhibits ABA-modulated chlorophyll accumulation
Three HIS-MdSnRK1.1 transgenic apple seedling lines were obtained for further study, and were identified at the gene expression level, the transcript level, and the protein level to ensure the accuracy of the experimental results (Fig. S3b-d, see online supplementary material).MdSnRK1.1-OE and GL-3 seedlings were treated with or without ABA to elucidate how MdSnRK1.1 responds to chlorophyll accumulation under ABA treatment.These results showed that MdSnRK1.1-OEplants were more sensitive to ABA than the wild type, with decreased chlorophyll content (Fig. 3a-c).Transcript levels of MdHEMA1, MdCAO, and MdCHLH decreased in MdSnRK1.1-OEplants after ABA treatment (Fig. 3d-f), suggesting that MdSnRK1.1 was involved in the reduction of chlorophyll content caused by ABA treatment.To further support this result, we performed the experiment that MdSnRK1.1 transgenic Arabidopsis treated with ABA.As a result, compared with Col, the MdSnRK1.1-OEArabidopsis seedlings gradually showed reduced biomass and chlorophyll content along with the decreased transcript levels of chloroplast developmental-related genes under ABA treatment (Fig. S4a-f, see online supplementary material).Therefore, these data indicated that MdSnRK1.1 regulated the ABA-modulated suppression of chlorophyll accumulation.In Arabidopsis, SnRK1 responds to ABA signaling pathway [9].The expression of the homologs genes in ABA signaling (including MdABI1 and MdABI2 belong to PP2Cs) [34] in apple were recused by ABA and the MdSnRK1.1 overexpression (Fig. S5a and b, see online supplementary material), and the transcripts of the overlapping genes of MdSnRK1.1 [including MDP0000173500 (MdSnRK1.2) and MDP0000320932 (MdSnRK1.3)][35] in apple were induced by ABA and the MdSnRK1.1 overexpression (Fig. S5c and d, see online supplementary material).

MdSnRK1.1 interacts with the MdGLK1 protein
In order to explore the molecular mechanism of MdSnRK1.1 participating in ABA-modulated chlorophyll accumulation, a Y2H screen was performed using MdSnRK1.1 as bait to identify any potentially interacting proteins.As a result, the chloroplast development related protein MdGLK1 was screened and used as a potential interaction protein for subsequent research.MdSnRK1.1 contained a typical catalytic subunit, and MdGLK1 contained a DNA binding domain.In order to determine the accurate interaction segment between MdSnRK1.1 and MdGLK1, we divided its sequence into several segments based on their structural domain.The MdSnRK1.1 gene was divided into two segments to  4a).Pull-down assays were carried out by using the MdSnRK1.1-GST,MdGLK1-HIS, and GST fusion proteins.These results showed that MdGLK1-HIS protein was enriched by MdSnRK1.1-GST.In contrast, the MdGLK1-HIS protein was not enriched by the GST control (Fig. 4b), indicating that MdSnRK1.1 physically interacted with MdGLK1.The Co-IP assay was performed to confirm MdSnRK1.1 interact with MdGLK1 in vivo (Fig. 4c).Then, a BiFC assay was conducted to further confirm this  4d).Lastly, dual luciferase assays were carried out in N. benthamiana leaves to confirm the interaction.The full-length sequences of MdSnRK1.1 and MdGLK1 were inserted into the pGreenII 0800-LUC vector to obtain the reporter constructs.Four combination vectors, including MdSnRK1.1-nLUC+MdGLK1-cLUC,MdSnRK1.1-nLUC+cLUC,nLUC+MdGLK1-cLUC, and nLUC+cLUC were injected into N. benthamiana leaves.As a result, only the MdSnRK1.1-nLUC+MdGLK1-cLUCtreatment group caused the N. benthamiana to f luoresce (Fig. 4e and f).Taken together, these data revealed that MdSnRK1.1 interacted with MdGLK1 in vivo and in vitro.

The chloroplast development gene MdGLK1 responds to ABA treatment
To test whether MdGLK1 involves in ABA-modulated chlorophyll accumulation, the expression level of MdGLK1 was first detected under ABA treatment.The qRT-PCR detection results showed that the expression of MdGLK1 was obviously repressed at 3 h after ABA treatment in apple (Fig. 5a).Then, glk mutant and glk/MdGLK1-OE transgenic Arabidopsis were obtained to verify the possible role of MdGLK1 in regulating ABA-modulated chlorophyll accumulation.When treated with ABA, the chlorophyll content of glk mutant plants was lower than that of Col, and the expression of chloroplast developmental-related genes were also significantly repressed (Fig. 5b-g).However, the complementation line rescued the phenotype of leaf chlorosis under ABA treatment (Fig. 5b and d).Compared to the wild-type, the transcript levels of the chloroplast developmental-related genes, including AtHEMA1, AtCAO, and AtCHLH, decreased in the glk mutant but increased

MdSnRK1.1 destabilizes the MdGLK1 protein
As the key metabolic switch, the SNF1 protein kinase acts on some enzymes and transcription factors and affects protein stability by phosphorylating its substrates in yeast [36][37][38].SNF1 responds to energy demand and environmental stress by inducing or repressing metabolic pathways [19].Considering the potential kinase activity of MdSnRK1.1 and the repressed expression of MdGLK1 under ABA conditions, we proposed that MdSnRK1.1 might affect the stability of MdGLK1 protein.An in vitro protein degradation assay was performed to explore the effect of MdSnRK1.1 on the protein stability of MdGLK1.The total protein extracts isolated from GL-3 with or without ABA treatment were cultured with the MdGLK1-HIS fusion protein.As a result, the MdGLK1-HIS protein level decreased significantly as ABA treatment time was extended (Fig. 6a and b).However, in the presence of MG132, the MdGLK1-HIS level was not affected by ABA treatment, which indicated the protein of MdGLK1-HIS is degraded in a 26S proteasome-dependent manner (Fig. 6a).These studies showed that ABA treatment significantly reduced the MdGLK1 protein content.To examine whether the overexpression  6c).The relative protein content of MdGLK1-HIS was 33% when treated with ABA for 5 h under the background of the GL-3 seedlings, while the relative protein content of MdGLK1-HIS was only 11% when treated with ABA for 5 h under the background of the HIS-MdSnRK1.1 transgenic apple seedlings (Fig. 6d).Furthermore, MdSnRK1.1-MdGLK1transgenic apple seedlings were obtained by instantaneous transformation.The in vivo protein degradation assay was carried out to identify the degradation of MdGLK1 was accelerated by MdSnRK1.1 under ABA treatment (Fig. S6, see online supplementary material).All of these results indicated that MdSnRK1.1 accelerated the degradation of MdGLK1 protein.

MdSnRK1.1 phosphorylates MdGLK1 and affects its protein stability
SnRK1 plays its role by phosphorylating downstream substrates under normal conditions [17].The protein phosphorylation process is often involved in the modulation of protein stability [39].To investigate whether MdSnRK1.1 affected the protein stability of MdGLK1 through phosphorylation, we first performed in vitro kinase assays that combined MdSnRK1.1-GSTkinase protein with the MdGLK1-HIS protein.A Phos-tag assay carried out that MdSnRK1.1 could phosphorylate MdGLK1 in vitro and this phosphorylation could be inhibited by phosphatase inhibitor cocktail (CIP) (Fig. 7a).To determine the potential phosphorylation site of MdGLK1, we logged on http://prosite.expasy.org/scanprosite to predict the possible phosphorylation sites of MdGLK1 according to the recognition sequence of

SnRK1 ([MLVFI]-[XRKH]-[XRKH]-XX-[ST]-XXX-[LFIMV]
).The three potential functional sites Ser468, Ser481, and Thr496 were analysed (Fig. 7b).An amino acid sequence alignment was constructed to determine the conservation of phosphorylation sites between the MdGLK1 with other GLK genes from Arabidopsis, rice, and maize.It was found that the serine at 468 of MdGLK1 was conserved in all species (Fig. 7c).Then, a variant of MdGLK1 harboring nonphosphorylatable Ser468 to Ala468 point mutation (S468A) was generated.In the gel containing Phos-tag, the phosphorylation of MdGLK1 was significantly weakened, but could not be diminished after the serine mutation at 468 (Fig. 7d).Meanwhile, the phosphorylation of MdGLK1 was not weakened after mutations at the Ser481 and Thr496 (Fig. S7, see online supplementary material).These results suggested S468 of MdGLK1 as target site of MdSnRK1.1 phosphorylation.To investigate the inf luence of MdSnRK1.1 on the stability of MdGLK1 protein after phosphorylation site mutation, the protein degradation assay was performed.The serine deletion at position 468 partially blocked the degradation of MdGLK1 induced by ABA treatment, suggesting that MdSnRK1.1 promoted the degradation of MdGLK1 protein through the phosphorylation pathway (Fig. 7e-f).To verify whether other post-translational modifications of proteins, such as ubiquitination, affect the degradation of MdGLK1 protein by MdSnRK1.1, we conducted a ubiquitination test and found that MdGLK1 did not undergo ubiquitination modification (Fig. S8, see online supplementary material).It is reported that GLK1 binds to conserved promoter sequences CCAATC of HEMA1 gene [31].EMSA analysis was conducted to test whether MdSnRK1.1 phosphorylation affected MdGLK1 binding to target gene MdHEMA1.MdGLK1 bound to the probe (−454) of MdHEMA1 promoter and the binding of MdGLK1 (S468A) to the MdHEMA1 promoter was significantly attenuated (Fig. S9, see online supplementary material).To verify whether phosphorylated MdGLK1 promotes or inhibits the expression of MdHEMA1 by binding to its promoter, we added a bimolecular luciferase assay.We found that the f luorescence signal of MdHEMA1pro:LUC + 35Spro:MdGLK1 (S468A) was comparable to that of MdHEMA1pro:LUC + 35Spro:62-SK, and its luciferase activity has not significantly increased, indicating that the mutation of MdGLK1 phosphorylation site inhibits its ability to activate downstream MdHEMA1 promoter transcription (Fig. S10, see online supplementary material).In general, MdSnRK1.1-mediatedphosphorylation was critical for MdGLK1 binding to the target gene promoters.

MdGLK1 and MdSnRK1.1 jointly regulate ABA-modulated chlorophyll accumulation
The tissue expression patterns revealed that MdSnRK1.1 and MdGLK1 had their highest expression levels in leaves (Fig. S11a  and b, see online supplementary material).The subcellular localization analysis of MdSnRK1.1 and MdGLK1 showed that MdSnRK1.1 was localized to the nucleus and cytoplasm, while MdGLK1 was localized to the nucleus (Fig. S11c, see online supplementary material).The genetic materials of MdGLK1-OE and MdSnRK1.1-OE/MdGLK1-OEplants were obtained to further explore the genetic regulation of MdSnRK1.1 and MdGLK1.These genetic materials treated with ABA showed that chlorophyll content accumulated in MdGLK1-OE under the ABA treatment, while it was reduced in MdSnRK1.1-OEplants compared to the Col (Fig. 8a).However, MdSnRK1.1-OE/MdGLK1-OE plants alleviated the MdSnRK1.1-OEyellow leaf phenotype (Fig. 8a-c), which was similar to the chlorophyll contents and the expression patterns of

Discussion
The chloroplast is an important place for plants to carry out photosynthesis, amino acid metabolism, hormone biosynthesis, and other metabolic processes [40][41][42].Chlorophyll is the most important pigment in plant photosynthesis.This is known as ABA is a stress hormone that induces leaf yellowing [2].In our study, we first discussed the relationship between ABA and chlorophyll.ABA treatment affects the content of chlorophyll in apple leaves, leading to the leaves of apple seedlings turning yellow (Fig. S1, see online supplementary material).Yellowing of leaves often indicates senescence in plants.It has been previously reported that ABA treatment accelerates the senescence process of plants [43][44][45].We therefore tested whether long-term ABA treatment induced leaf yellowing by promoting leaf senescence, thus clarifying the molecular mechanism of ABA-induced leaf yellowing (Fig. S2, see online supplementary material).We found that DMSO restored the leaf yellowing phenotype caused by ABA treatment.However, plant aging is irreversible [46].We demonstrated that the treatment of ABA induced leaf yellowing by inhibiting the accumulation of chlorophyll in apple.
The ABA signal response is a complex process involving a number of genes, such as PYR, PP2Cs, and SnRK1 [3,4].In Arabidopsis, SnRK1, as an important protein kinase, participates in ABA signaling through its interaction with PP2C [9].Overexpression of AtSnRK1.1 in Arabidopsis delays the growth of seedlings and enhances the response to ABA.However, the overexpression of AtSnRK1.1 had no effect on Arabidopsis seed germination [18].It has been reported that SnRK1 increases ABA levels in pea embryos, thereby regulating ABA-mediated seed maturation [47].However, the SnRK1 overexpression is not to alter the ABA level in Arabidopsis [18].Overexpression of MdSnRK1.1 promotes maturation of tomato fruit, and ABA plays a role in tomato ripening [48].We found that ABA increased the MdSnRK1.1 transcription level (Fig. 2).Previous studies have shown that MdSnRK1.1-OEapple calli leads to a highly ABA-sensitive phenotype, suggesting that MdSnRK1.1 participates in the ABA signaling pathway in apple [24].We verified that ABA-induced leaf yellowing of MdSnRK1.1-OEseedlings was related to chlorophyll accumulation (Fig. 3).ABA-induced leaf yellowing, and overexpression of MdSnRK1.1 aggravated this phenomenon (Fig. 3).Due to plants being unable to move, they must have various ways to cope with the constantly changing external environmental conditions.Therefore, MdSnRK1.1 plays a balanced role in ABA-mediated growth inhibition and chloroplast development-induced growth promotion.In addition, SnRK1 directly destabilizes the ETHYLENE INSENSI-TIVE3 (EIN3) to slow down senescence-associated leaf yellowing in Arabidopsis [49].We speculated that SnRK1 jointly regulated the leaf yellowing phenotype through two hormone pathways.
In this study, we proposed a new mechanism by which MdSnRK1.1 participates in ABA-mediated leaf chlorosis through its interaction with chloroplast developmental-related protein MdGLK1 (Fig. 4).MdGLK1 was expressed in photosynthetic tissues, such as leaves, which is the same location as MdSnRK1.1 (Fig. S8, see online supplementary material).GLK complements the Arabidopsis glk1glk2 double mutant, in which chloroplasts exhibit an increase in the number of thylakoid grana [50].Here, ABA negatively regulated the expression of MdGLK1 and accelerated degradation of the MdGLK1 protein (Figs 5a and 6a).Overexpressing MdGLK1 attenuated the ABA-induced yellowing leaf phenotype in Col and MdSnRK1.1-OEArabidopsis seedlings (Fig. 8a).These findings described that MdSnRK1.1 and MdGLK1 suppressed chlorophyll accumulation under ABA treatment.
More studies are needed to understand the relationship between MdSnRK1.1 and MdGLK1.SnRK1 is very conservative in evolution as a protein kinase.SnRK1 forms heterotrimeric complexes with the catalytic α-subunit and regulatory β and γ -subunits [10,39,51].SnRK1 affects the stability of downstream kinases or transcription factors through pathways, such as phosphorylation [24,36].Stability of the MdGLK1 protein was affected by ABA treatment.MdGLK1 was degraded under the ABA treatment, which is consistent with the leaf yellowing caused by ABA when chlorophyll accumulation was suppressed.The protein degradation assay demonstrated that the MdGLK1 protein was less stable under the MdSnRK1.1-OEseedling condition than under the control condition after ABA treatment (Fig. 6).All of these data indicated that MdSnRK1.1 was critical for the degradation of MdGLK1, and MdSnRK1.1 inhibited the activity of MdGLK1 to induce suppression of chlorophyll accumulation under an ABA treatment.
Considering the involvement of MdGLK1 in the posttranslational modification process to the protein, we speculated that it might undergo ubiquitination degradation dependent on the 26S proteasome pathway.The ubiquitination test found that MdGLK1 did not undergo ubiquitination modification (Fig. S9, see online supplementary material).In Arabidopsis, SnRK1 phosphorylates its specific substrates to be involved in various life processes [21].The substrates include the key metabolic enzymes (such as sucrose phosphate synthase, nitrate reductase, and 3hydroxy-3-meth-ylglutaryl CoA reductase), and the transcription factors (such as FUSCA3) [52][53][54].AtSnRK1.1 phosphorylates and positively regulates FUSCA3 to modulate ABA responses [54,55].Previous studies reported that MdSnRK1.1 interacted with and phosphorylated MdCAIP1 in apple [24].Meanwhile, we screened Ser468 of MdGLK1 as a potential target site for MdSnRK1.1 phosphorylation, and this phosphorylation site is crucial for the protein degradation of MdGLK1 by MdSnRK1.1 and the binding of MdGLK1 to the MdHEMA1 gene promotor.Overall, our findings suggested that MdSnRK1.1 phosphorylated MdGLK1 and accelerated its degradation to involve the ABA signaling pathway, providing a regulatory mechanism for further study of ABA signaling.

Conlusion
Taken together, our data provide a mechanism of the ABAmodulated chlorophyll accumulation in apple (Fig. 9).MdSnRK1.1 represses the ABA-regulated chlorophyll accumulation by direct protein-protein interaction with MdGLK1 and inhibits the transcriptional activation of downstream target gene MdHEMA1 by MdGLK1.Under ABA deficiency conditions, the transcription level of MdSnRK1.1 is reduced, and the phosphorylation of MdSnRK1.1 on MdGLK1 is weakened, which contributes to chlorophyll accumulation.In the condition of ABA, MdSnRK1.1 contributes to the degradation of the MdGLK1 protein via the 26S proteasome pathway, inhibiting the transcriptional activation of the MdHEMA1 by MdGLK1, thereby inhibiting the accumulation of chlorophyll.

Acquisition of transgenic plants
The open reading frames (ORFs) of the MdSnRK1.1 and MdGLK1 genes were cloned into the pRI101 vector.The overexpressing plasmids were constructed by using Agrobacterium (LBA4404)mediated transformation that transformed the MdSnRK1.1-pRIrecombinant plasmid into 'GL-3' cultures [57].The transgenic lines were selected on MS medium that contained 250 mg/L timentin, 0.5 mg/L NAA, 2 mg/L thidiazuron (TDZ), and 25 mg/L kanamycin for apple seedlings for 30 days under a dark period.For Arabidopsis transformation, the vectors were genetically transformed into Col with Agrobacterium tumefaciens strain GV3101 using the f loral dip transformation method [58], and the third generation of transformed Arabidopsis plants was analysed.For N. benthamiana agrobacterium-mediated infiltration, the vectors were genetically transformed into GV3101 until the culture grew to saturation.The precipitate was mixed with the solution containing 100 mM acetosyringone, 25 mM MgCl 2 , and 25 mM MES and kept at 25 • C for 4 h, and then injected into N. benthamiana leaves using a needleless syringe.

Quantitative real-time polymerase chain reaction (qRT-PCR)
The RNAplant plus Reagent (Tiangen, Beijing, China) system was used to extract the total RNA that was isolated from apple and Arabidopsis plants.The PrimeScript First Strand cDNA Synthesis Kit (Takara, Beijing, China) was used to compose the first-strand cDNA.The iCycler iQ5 Detection System (Bio-Rad, Hercules, CA, USA) was used to conduct the qRT-PCR reactions using the SYBR Green method.The 18S in apple was used as the loading control and each operation was performed as technical replicates three times [59].All primers used in this study are presented in Table S1 (see online supplementary material).

Measurement of chlorophyll content
A total of 1 g of Arabidopsis or apple seedling leaves was dipped into 95% ethanol in the dark for 1 day at 24 • C to extract chlorophyll.A spectrophotometer was used to colorimetrically calculate the absorbance at 649 nm and 665 nm when the leaves turned white.Chlorophyll content (mg/g FW) = (20.8× A645 + 8.04 × A663) × V/M.(A: absorbance; V: volume of alcohol; M: weight of leaves).

Analysis of subcellular localization
The MdSnRK1.1 and MdGLK1 coding sequences were contracted into the green f luorescent protein (GFP) vector.The 35S::GFP-MdSnRK1.1, 35S::GFP-MdGLK1, and the 35S:GFP were transferred into the GV3101 strain.The expression vectors were infiltrated into N. benthamiana cells using the Agrobacterium-mediated method and grown for 4 days.The expression of the GFP f luorescent protein in the cells was captured by a high-resolution confocal microscope (LSCM; Carl Zeiss, Oberkochen, Germany).

Protein degradation assay
The method of protein degradation assay was based on the paper published by An et al. [57].The in vitro protein was induced by 0.3 mM isopropyl β-D-thiogalactoside (IPTG) from E. coil cells for 5 h at 37 • C. The in vivo proteins were extracted from the transgenic apple seedlings in degradation buffer containing 2 mM phenylmethylsulfonylf luoride (PMSF), 4 mM DTT, 8 mM NaCl, 12 mM MgCl 2 , 15 mM ATP, and 25 mM Tris-HCl.The mixed solutions were detected at 0, 1, 3, and 5 h, and used HIS, GFP, or ACTIN as detection antibody.

Dual luciferase assay
The ORFs of MdSnRK1.1 and MdGLK1 were transferred into the pGreenII 0800-nLUC vector and the pGreenII 0800-cLUC vector, respectively, to contract the MdSnRK1.1-nLUC and MdGLK1-cLUC.The mixture of MdSnRK1.1-nLUC,MdGLK1-cLUC, nLUC, and cLUC were inserted into N. benthamiana cells.After 3 days, a live-cell image was used to measure luminescence.Detection of the LUC/REN activity was with the luciferase reporter gene detection kit (Sigma, Shanghai, China).

Pull-down assay
The full-lengths of MdSnRK1.1 and MdGLK1 cDNA were transferred into the pGEX-4 T and pET-32a vector, respectively.These plasmids were transformed into Escherichia coli BL21 (DE3).Magnetic bead method was used to mix the induced fusion proteins, and the precipitate was detected with HIS or GST antibodies (Abmart, Shanghai, China).

Bimolecular fluorescence complementation (BiFC) assay
The sequence of MdSnRK1.1 was inserted to the C-terminal half of the yellow f luorescent protein (cYFP) to contract the MdSnRK1.1-cYFP and the sequence of MdGLK1 was inserted to the N-terminal half of YFP to contract the MdGLK1-nYFP.These recombinant plasmids were inserted into the N. benthamiana epidermal cells, then grown for 3 days.The signal of YFP f luorescent protein in cells was captured by LSCM.

In vitro phosphorylation assay
The MdGLK1-HIS protein and the MdSnRK1.1-GSTkinase protein were cultured in a 20 ml reaction buffer containing 1 mM DTT, 8 mM MgCl 2 , 25 mM ATP, and 50 mM Tris-HCl.The solution was cultured at 30 • C for 2 h and terminated by adding protein loading buffer.Subsequently, the samples were separated on 10% Phostag SDS-PAGE gels supplied with 100 μM Phos-tag (Nard, Shanghai, China) and 50 mM MnCl 2 , and detected with HIS or GST antibodies (Abmart, Shanghai, China).

Electrophoretic mobility shift assay (EMSA)
The glutathione sepharose beads (Thermo Fisher, Scientific, Waltham, MA, USA) were used to purify the MdGLK1-HIS protein.
The EMSA probe biotin labeling kit (Beyotime, Shanghai, China) was used to label the probe of the MdHEMA1 promoter.The competitors are the same sequences without labels.The purified protein and labeled probe were mixed at 25 • C for 40 min and separated on nondenaturing polyacrylamide gels.

Figure 1 .
Figure 1.Inhibition of chlorophyll accumulation under ABA treatment.a Phenotypes of GL-3 apple seedling leaves in the presence or absence of 150 μM ABA for 15 days.Scale bars, 1 cm.b Chlorophyll contents of GL-3 apple seedling leaves treated with or without ABA for 15 days.c-e Relative expression of MdHEMA1, MdCAO, and MdCHLH in GL-3 apple seedling leaves treated with or without ABA for 15 days.The expression levels before the treatment (DMSO, 0 days) might be set to 1. Values are mean ± SD of three biological replicate experiments and asterisks denote a significant difference compared to the control: * P < 0.05.
propose a potential model to describe the role of MdSnRK1.1.in the modulation of ABA response.The protein kinase MdSnRK1.1 participated in the apple ABA signaling pathway by interacting with MdGLK1 which is a chloroplast development-related protein.MdSnRK1.1 phosphorylated MdGLK1 and reduced its protein stability.Accordingly, this study uncovered the mechanism of how MdSnRK1.1-MdGLK1regulates ABA-modulated chlorophyll accumulation in apple.

Figure 3 .
Figure 3. MdSnRK1.1 plays an essential role in ABA-induced leaf yellowing.a, b Phenotypes of the WT and MdSnRK1.1-OEapple seedlings under the ABA treatment.Scale bars, 1 cm.c Chlorophyll contents of the WT and MdSnRK1.1-OEapple seedlings under the ABA treatment.d-f Relative expression of MdHEMA1, MdCAO, and MdCHLH in WT and MdSnRK1.1-OEapple seedling plants after treated with ABA.The expression levels in WT treated with DMSO might be set to 1. Values are mean ± SD of three biological replicate experiments and asterisks denote significant differences compared to the control: * P < 0.05; * * P < 0.01.

Figure 4 .
Figure 4. MdSnRK1.1 interacts with the MdGLK1 protein.a Yeast two-hybrid assay to examine the interaction between MdSnRK1.1 and MdGLK1.Scale bars, 5 mm.b In vitro pull-down assay indicating the interaction between MdSnRK1.1 and MdGLK1.c Co-IP assay indicating the interaction between MdSnRK1.1 and MdGLK1.d BiFC assay in Nicotiana benthamiana leaves showing the interaction between MdSnRK1.1 and MdGLK1.Scale bars, 50 μm.e-f The dual luciferase assay was performed in N. benthamiana leaves to verify the interaction between MdSnRK1.1-nLUC and MdGLK1-cLUC.Scale bars, 1 cm.

Figure 5 .
Figure 5. MdGLK1 is involved in ABA-induced leaf yellowing.a The expression of MdGLK1 by qRT-PCR in GL-3 seedlings treated with ABA.The expression levels before the treatment (DMSO, 0 hours) might be set to 1. b Phenotypes of the glk mutant and the glk/MdGLK1-OE Arabidopsis complementation line.Scale bars, 2 cm.c Fresh weight of the glk mutant and the glk/MdGLK1-OE Arabidopsis complementation line.d Chlorophyll contents of the glk mutant and the glk/MdGLK1-OE Arabidopsis complementation line.e-g Relative expression of AtHEMA1, AtCAO, and AtCHLH in the glk mutant and glk/MdGLK1-OE Arabidopsis complementation line.The expression levels in Col treated with DMSO might be set to 1. Values are mean ± SD of three biological replicate experiments and asterisks denote significant differences compared to the control: * P < 0.05; * * P < 0.01.

Figure 6 .
Figure 6.MdSnRK1.1 promotes destabilization of MdGLK1 protein.a The MdGLK1-HIS protein and total protein samples were extracted from GL-3 apple seedlings treated with or without ABA and incubated together at 22 • C for the indicated period.c The MdGLK1-HIS protein and total protein samples were extracted from MdSnRK1.1-OE apple seedlings treated with or without ABA and incubated together at 22 • C for the indicated period.b, d The protein levels at 0, 1, 3, and 5 h were examined in a and c.

Figure 7 .
Figure 7. MdSnRK1.1 affects the stability of MdGLK1 protein by phosphorylating serine at 468 position of MdGLK1.a Modulation of MdGLK1 phosphorylation by MdSnRK1.1 in vitro.b Analysis of three potential phosphorylation sites predicted by the website.c Conservative analysis of potential phosphorylation sites in various species.d Modulation of serine 468 mutation MdGLK1 phosphorylation by MdSnRK1.1 in vitro.e The MdGLK1-HIS protein or MdGLK1(S468A)-HIS mixed with total protein samples which extracted from GL-3 apple seedlings treated with or without ABA and incubated together at 22 • C for the indicated period.f The nomalized band intensity were examined in e.

Figure 9 .
Figure 9.A model of MdSnRK1.1-mediatedchlorophyll accumulation in response to ABA in apple.