Apple U-box-type E3 ubiquitin ligase MdPUB23 reduces cold-stress tolerance by degrading the cold-stress regulatory protein MdICE1.

Cold stress limits plant growth, geographical distribution, and crop yield. The MYC-type bHLH transcription factor ICE1 is recognized as the core positive regulator of the cold-stress response. However, how ICE1 protein levels are regulated remains to be further studied. In this study, we observed that a U-box-type E3 ubiquitin ligase, MdPUB23, positively regulated the cold-stress response in apple. The expression of MdPUB23 increased at both the transcriptional and post-translational levels in response to cold stress. Overexpression of MdPUB23 in transgenic apple enhanced sensitivity to cold stress. Further study showed that MdPUB23 directly interacted with MdICE1, promoting the ubiquitination-mediated degradation of the MdICE1 protein through the 26S-proteasome pathway and reducing the MdICE1-improved cold-stress tolerance in apple. Our results reveal that MdPUB23 regulates the cold-stress response by directly mediating the stability of the positive regulator MdICE1. The PUB23-ICE1 ubiquitination module may play a role in maintaining ICE1 protein homeostasis and preventing overreactions from causing damage to plants. The discovery of the ubiquitination regulatory pathway of ICE1 provides insights for the further exploration of plant cold-stress-response mechanisms.

Apple SINA E3 ligase MdSINA3 negatively mediates JA-triggered leaf senescence by ubiquitinating and degrading the MdBBX37 protein.

Jasmonic acid (JA) induces chlorophyll degradation and leaf senescence. B-box (BBX) proteins play important roles in the modulation of leaf senescence, but the molecular mechanism of BBX protein-mediated leaf senescence remains to be further studied. Here, we identified the BBX protein MdBBX37 as a positive regulator of JA-induced leaf senescence in Malus domestica (apple). Further studies showed that MdBBX37 interacted with the senescence regulatory protein MdbHLH93 to enhance its transcriptional activation on the senescence-associated gene MdSAG18, thereby promoting leaf senescence. Moreover, the JA signaling repressor MdJAZ2 interacted with MdBBX37 and interfered with the interaction between MdBBX37 and MdbHLH93, thereby negatively mediating MdBBX37-promoted leaf senescence. In addition, the E3 ubiquitin ligase MdSINA3 delayed MdBBX37-promoted leaf senescence through targeting MdBBX37 for degradation. The MdJAZ2-MdBBX37-MdbHLH93-MdSAG18 and MdSINA3-MdBBX37 modules realized the precise modulation of JA on leaf senescence. In parallel, our data demonstrate that MdBBX37 was involved in abscisic acid (ABA)- and ethylene-mediated leaf senescence through interacting with the ABA signaling regulatory protein MdABI5 and ethylene signaling regulatory protein MdEIL1, respectively. Taken together, our results not only reveal the role of MdBBX37 as an integration node in JA-, ABA- and ethylene-mediated leaf senescence, but also provide new insights into the post-translational modification of BBX proteins.

The SUMO E3 Ligase MdSIZ1 Sumoylates a Cell Number Regulator MdCNR8 to Control Organ Size.

Plant growth and organ size putatively associated with crop yield are regulated by a complex network of genes including ones for controlling cell proliferation. The gene fw2.2 was first identified in tomatoes and reported to govern fruit size variation through controlling cell division. In this study, we isolated a putative ortholog of the tomato fw2.2 gene from apple, Cell Number Regulator 8 (MdCNR8). Our functional analysis showed that MdCNR8 may control fruit size and root growth. MdCNR8 was mediated by the SUMO E3 ligase MdSIZ1, and SUMOylation of MdCNR8 at residue-Lys39 promoted the translocation of MdCNR8 from plasma membrane to the nucleus. The effect of MdCNR8 in inhibiting root elongation could be completely counteracted by the coexpression of MdSIZ1. Moreover, the lower cell proliferation of apple calli due to silencing MdSIZ1 could be rescued by silencing MdCNR8. Collectively, our results showed that the MdSIZ1-mediated SUMOylation is required for the fulfillment of MdCNR8 in regulating cell proliferation to control plant organ size. This regulatory interaction between MdSIZ1 and MdCNR8 will facilitate understanding the mechanism underlying the regulation of organ size.

Organization and regulation of the apple SUMOylation system under salt and ABA.

Small ubiquitin-related modifier (SUMO)-mediated post-translational protein modification is widely conserved among eukaryotes. SUMOylation refers to the covalent attachment of SUMO to target proteins that alters their function, location, and protein-protein interactions when plants are under abiotic stress. We identified 37 genes in the apple genome that encoded members of the SUMOylation pathway. In addition, RNA-Seq data shows their expression levels between different tissues. We can find that there are mainly expressed genes between each component to ensure that the entire pathway works in the plant. We found that the expression levels of 12 genes were significantly changed under NaCl and ABA treatment through qRT-PCR. MdSIZ1a strongly expression responded to NaCl and ABA treatment. Subsequently, MdSIZ1a was cloned and transformed into apple callus, further verifying the important role of the SUMOylation pathway under stress conditions. The interaction between MdSIZ1a and MdSCEa was verified by yeast two-hybrid, confirming that MdSIZ1a acts as bridge enzyme on MdSCEa and target substrates. Finally, we predicted and analyzed the functional interaction network of E3 ligase to shed light on protein interactions and gene regulatory networks associated with DNA damage repair under abiotic stress in apples.

Phytochrome interacting factor MdPIF7 modulates anthocyanin biosynthesis and hypocotyl growth in apple.

Light affects many physiological and developmental processes of plants by regulating the expression and activity of light-responsive proteins. Among them, phytochrome interacting factors (PIFs) play pivotal roles in the regulation of anthocyanin accumulation and hypocotyl growth. However, the molecular mechanism is not well understood, especially in woody plants, such as apple (Malus × domestica). In this study, we identified a light-responsive PIF protein, MdPIF7, in apple and investigated the molecular mechanism of its regulation of anthocyanin biosynthesis and hypocotyl growth. We found that overexpression of MdPIF7 decreased anthocyanin accumulation in transgenic apple materials and promoted hypocotyl elongation in ectopically expressed Arabidopsis (Arabidopsis thaliana). Further investigation showed that MdPIF7 functioned by interacting with B-box 23 (MdBBX23), a positive regulator of anthocyanin biosynthesis in apple and hypocotyl growth inhibition in ectopically expressed Arabidopsis, and attenuating the transcriptional activation of MdBBX23 on LONG HYPOCOTYL 5 (MdHY5). In addition, MdPIF7 interacted with basic region leucine zipper 44 (MdbZIP44) and ethylene response factor 38 (MdERF38), two positive regulators of anthocyanin biosynthesis, and it negatively regulated MdbZIP44- and MdERF38-promoted anthocyanin accumulation by interfering with the interaction between MdbZIP44/MdERF38 and MdMYB1. Taken together, our results reveal that MdPIF7 regulates anthocyanin biosynthesis in apple and hypocotyl growth in ectopically expressed Arabidopsis through MdPIF7-MdBBX23-MdHY5 and MdPIF7-MdbZIP44/MdERF38-MdMYB1 modules. Our findings enrich the functional studies of PIF proteins and provide insights into the molecular mechanism of PIF-mediated anthocyanin biosynthesis and hypocotyl growth.

Abscisic acid insensitive 4 interacts with ICE1 and JAZ proteins to regulate ABA signaling-mediated cold tolerance in apple.

Abscisic acid is involved in the regulation of cold stress response, but its molecular mechanism remains to be elucidated. In this study, we demonstrated that the APETALA2/ethylene responsive factor (AP2/ERF) family protein MdABI4 positively regulates abscisic acid-mediated cold tolerance in apple. We found that MdABI4 interacts with MdICE1, a key regulatory protein involved in the cold stress response, and enhances the transcriptional regulatory function of MdICE1 on its downstream target gene MdCBF1, thus improving abscisic acid-mediated cold tolerance. The jasmonate-ZIM domain (JAZ) proteins MdJAZ1 and MdJAZ2 negatively modulate MdABI4-improved cold tolerance in apple by interacting with the MdABI4 protein. Further investigation showed that MdJAZ1 and MdJAZ2 interfere with the interaction between the MdABI4 and MdICE1 proteins. Together, our data revealed that MdABI4 integrates jasmonic acid and abscisic acid signals to precisely modulate cold tolerance in apple through the JAZ-ABI4-ICE1-CBF regulatory cascade. These findings provide insights into the crosstalk between jasmonic acid and abscisic acid signals in response to cold stress.

Apple SUMO E3 ligase MdSIZ1 facilitates SUMOylation of MdARF8 to regulate lateral root formation.

Post-translational modification of proteins mediated by SIZ1, a small ubiquitin-like modifier (SUMO) E3 ligase, regulates multiple biological processes in plants. However, its role in the regulation of lateral root formation remains unclear. Here, we demonstrate that the apple SUMO E3 ligase MdSIZ1 promotes lateral root formation. Using a yeast-two-hybrid (Y2H) system, the auxin response factor MdARF8 was screened out as a protein-protein interaction partner of the SUMO-conjugating E2 enzyme MdSCE1, indicating that MdARF8 may be a substrate for MdSIZ1. The interaction between MdARF8 and MdSCE1 was confirmed by pull-down, Y2H and Co-immunoprecipitation assays. MdSIZ1 enhanced the conjugating enzyme activity of MdSCE1 to form a MdSCE1-MdSIZ1-MdARF8 complex, thereby facilitating SUMO modification. We identified two arginine substitution mutations at K342 and K380 in MdARF8 that blocked MdSIZ1-mediated SUMOylation, indicating that K342 and K380 are the principal SUMOylation sites of the MdARF8 protein. Moreover, MdARF8 promoted lateral root formation in transgenic apple plants, and the phenotype of reduced lateral roots in the Arabidopsis siz1-2 mutant was restored in siz1-2/MdARF8 complementary plants. Our findings reveal an important role for sumoylation in the regulation of lateral root formation in plants.

An apple long-chain acyl-CoA synthetase, MdLACS4, induces early flowering and enhances abiotic stress resistance in Arabidopsis.

The aerial parts of apple are protected against environmental stress by cuticular wax. Although it has been suggested that several long-chain acyl-CoA synthetases are involved in wax biosynthesis, the molecular pathway of apple cuticular wax biosynthesis remains unclear. In this study, an MdLACS4 protein with long-chain acyl-CoA synthetase activity was isolated from apple. The MdLACS4 gene was highly expressed in pericarp, stem, and mature leaf tissues. Ectopic expression of MdLACS4 in Arabidopsis induced early flowering. Compared with wild-type plants, MdLACS4 transgenic Arabidopsis exhibited lower water loss rates, reduced epidermal permeability, increased cuticular wax in stems and leaves, and altered cuticular ultrastructure. Furthermore, the accumulation of cuticular wax enhanced the resistance of MdLACS4 transgenic plants to drought and salt stress. Finally, predicted protein functional interaction networks for LACS4 suggested that the molecular regulation pathway of MdLACS4 mediates wax biosynthesis in apple.

An apple long-chain acyl-CoA synthetase 2 gene enhances plant resistance to abiotic stress by regulating the accumulation of cuticular wax.

Apple cuticular wax can protect plants from environmental stress, determine fruit luster and improve postharvest fruit storage quality. In recent years, dry weather, soil salinization and adverse environmental conditions have led to declines in apple fruit quality. However, few studies have reported the molecular mechanisms of apple cuticular wax biosynthesis. In this study, we identified a long-chain acyl-CoA synthetase MdLACS2 gene from apple. The MdLACS2 protein contained an AMP-binding domain and demonstrated long-chain acyl-CoA synthetase activity. MdLACS2 transgenic Arabidopsis exhibited reductions in epidermal permeability and water loss; change in the expression of genes related to cuticular wax biosynthesis, transport and transcriptional regulation; and differences in the composition and ultrastructure of cuticular wax. Moreover, the accumulation of cuticular wax enhanced the resistance of MdLACS2 transgenic plants to drought and salt stress. The main protein functional interaction networks of LACS2 were predicted, revealing a preliminary molecular regulation pathway for MdLACS2-mediated wax biosynthesis in apple. Our study provides candidate genes for breeding apple varieties and rootstocks with better fruit quality and higher stress resistance.

The R2R3 MYB transcription factor MdMYB30 modulates plant resistance against pathogens by regulating cuticular wax biosynthesis.

BACKGROUND: The MYB transcription factor family is one of the largest transcriptional factor families in plants and plays a multifaceted role in plant growth and development. However, MYB transcription factors involved in pathogen resistance in apple remain poorly understood. RESULTS: We identified a new MYB family member from apple, and named it MdMYB30. MdMYB30 was localized to the nucleus, and was highly expressed in young apple leaves. Transcription of MdMYB30 was induced by abiotic stressors, such as polyethylene glycol and abscisic acid. Scanning electron microscopy and gas chromatograph-mass spectrometry analyses demonstrated that ectopically expressing MdMYB30 in Arabidopsis changed the wax content, the number of wax crystals, and the transcription of wax-related genes. MdMYB30 bound to the MdKCS1 promoter to activate its expression and regulate wax biosynthesis. MdMYB30 also contributed to plant surface properties and increased resistance to the bacterial strain Pst DC3000. Furthermore, a virus-based transformation in apple fruits and transgenic apple calli demonstrated that MdMYB30 increased resistance to Botryosphaeria dothidea. Our findings suggest that MdMYB30 plays a vital role in the accumulation of cuticular wax and enhances disease resistance in apple. CONCLUSIONS: MdMYB30 bound to the MdKCS1 gene promoter to activate its transcription and regulate cuticular wax content and composition, which influenced the surface properties and expression of pathogenesis-related genes to resistance against pathogens. MdMYB30 appears to be a crucial element in the formation of the plant cuticle and confers apple with a tolerance to pathogens.

Apple AP2/EREBP transcription factor MdSHINE2 confers drought resistance by regulating wax biosynthesis.

MAIN CONCLUSION: This study showed that AP2/EREBP transcription factor MdSHINE2 functioned in mediating cuticular permeability, sensitivity to abscisic acid (ABA), and drought resistance by regulating wax biosynthesis. Plant cuticular wax plays crucial roles in protecting plants from environmental stresses, particularly drought stress. Many enzymes and transcription factors involved in wax biosynthesis have been identified in plant species. In this study, we identified an AP2/EREBP transcription factor, MdSHINE2 from apple, which is a homolog of AtSHINE2 in Arabidopsis. MdSHINE2 was constitutively expressed at different levels in various apple tissues, and the transcription level of MdSHINE2 was induced substantially by abiotic stress and hormone treatments. MdSHINE2-overexpressing Arabidopsis exhibited great change in cuticular wax crystal numbers and morphology and wax composition of leaves and stems. Moreover, MdSHINE2 heavily influenced cuticular permeability, sensitivity to abscisic acid, and drought resistance.

The SUMO E3 Ligase MdSIZ1 Targets MdbHLH104 to Regulate Plasma Membrane H+-ATPase Activity and Iron Homeostasis.

SIZ1 (a SIZ/PIAS-type SUMO E3 ligase)-mediated small ubiquitin-like modifier (SUMO) modification of target proteins is important for various biological processes related to abiotic stress resistance in plants; however, little is known about its role in resistance toward iron (Fe) deficiency. Here, the SUMO E3 ligase MdSIZ1 was shown to be involved in the plasma membrane (PM) H+-ATPase-mediated response to Fe deficiency. Subsequently, a basic helix-loop-helix transcription factor, MdbHLH104 (a homolog of Arabidopsis bHLH104 in apple), which acts as a key component in regulating PM H+-ATPase-mediated rhizosphere acidification and Fe uptake in apples (Malus domestica), was identified as a direct target of MdSIZ1. MdSIZ1 directly sumoylated MdbHLH104 both in vitro and in vivo, especially under conditions of Fe deficiency, and this sumoylation was required for MdbHLH104 protein stability. Double substitution of K139R and K153R in MdbHLH104 blocked MdSIZ1-mediated sumoylation in vitro and in vivo, indicating that the K139 and K153 residues were the principal sites of SUMO conjugation. Moreover, the transcript level of the MdSIZ1 gene was substantially induced following Fe deficiency. MdSIZ1 overexpression exerted a positive influence on PM H+-ATPase-mediated rhizosphere acidification and Fe uptake. Our findings reveal an important role for sumoylation in the regulation of PM H+-ATPase-mediated rhizosphere acidification and Fe uptake during Fe deficiency in plants.

Genome-wide identification and characterization of apple long-chain Acyl-CoA synthetases and expression analysis under different stresses.

Long-chain acyl-CoA synthetases (LACSs) are members of the acyl-activating enzyme superfamily that have important roles in lipid synthesis and storage, fatty acid catabolism, vectorial acylation, and synthesis of cutin and wax. Here, 11 apple MdLACS genes were identified based on the Malus × domestica reference genome, clustered into six groups and mapped to ten chromosomes. Multiple sequence alignment and conserved motifs analyses showed that the sequences of the AtLACS and MdLACS proteins were highly conserved. A cis-element analysis in the promoter regions of the MdLACS genes revealed various elements related to stress responsiveness and plant hormones. Subsequently, expression analysis demonstrated that the MdLACS genes had different expression profiles in different tissues in response to various abiotic stresses. To further study the function of MdLACS genes in apple, MdLACS1 was isolated to identify its basic function, which the function of MdLACS1 in response to apple abiotic stress resistance was determined by the transgenic method. The results showed the MdLACS1 enhanced tolerance to polyethylene glycol, salt, and abscisic acid in the apple callus, suggesting that MdLACS1 is an important regulator in response to abiotic stresses. Finally, the functional interoperability network among the MdLACS proteins was predicted and analyzed, which could the understanding of the possible interactions among proteins and genes regulatory networks concerned with wax biosynthesis and regulatory mechanisms in response to abiotic stresses in apple.