Bench-grafted 'Fuji/M.26' apple (Malus domestica Borkh.) trees received a constant nitrogen (N) supply (10.7 mM) from bud break to the end of June, and were then fertigated with 0, 5, 10, 15 or 20 mM N in a modified Hoagland's solution for 2 months during the summer. In mid-October, half of the trees fertigated at each N concentration were sprayed twice with 3% urea, whereas the remaining trees served as controls. All trees were harvested after natural leaf fall and were stored at 2 degrees C. Five trees from each of the N treatment combinations were destructively sampled during dormancy to determine the composition of N and total nonstructural carbohydrates (TNC). As the N supply from fertigation increased, amounts of N in both free amino acids and proteins increased, whereas C/N ratios decreased. Foliar urea applications in the fall significantly increased amounts of N in both free amino acids and proteins, but decreased their C/N ratios. Arginine, the most abundant amino acid in both free amino acids and in proteins, accounted for an increasing proportion of N in free amino acids and proteins with increasing N supply from fertigation or foliar urea application. The ratio of protein N to free amino acid N decreased from about 27.1 to 3.2 as N supply from fertigation increased from 0 to 20 mM, and decreased further to 3.0 in response to foliar urea applications in the fall. Concentrations of glucose, fructose, sucrose and TNC decreased as the N supply from fertigation increased, whereas concentrations of sorbitol and starch remained relatively unchanged. Foliar urea applications decreased the concentration of each TNC component and the TNC concentration in each N fertigation treatment. A negative linear relationship was found between carbon in TNC and N in proteins and free amino acids. The sum of carbon in TNC, proteins and free amino acids remained constant in response to N supply from fertigation. However, foliar urea applications decreased the sum of carbon in proteins, free amino acids and TNC because about 21% of the decrease in TNC carbon was not recovered in free amino acids or proteins. Young apple trees store N and carbon dynamically in response to N supply. As N supply increases, an increasing proportion of N is found in the form of free amino acids, which have a low carbon cost, although proteins remain the main form of N storage. Furthermore, part of the carbon from TNC is incorporated into amino acids and proteins, decreasing the carbon stored as TNC and increasing the carbon stored as amino acids and proteins.

Low temperature affects the yield and quality of crops. Inducer of CBF expression 1 (ICE1) plays a positive role in plant cold tolerance by promoting the expression of CRT binding factor (CBF) and cold-responsive (COR) genes. Several ICE1-interacting transcription factors (TFs) that regulate plant cold tolerance have been identified. However, how these TFs affect the function of ICE1 and CBF expression under cold conditions remains unclear. Here, we identified the MYC-type TF MdbHLH4, a negative regulator of cold tolerance in Arabidopsis (Arabidopsis thaliana) and apple (Malus domestica) plants. Under cold conditions, MdbHLH4 inhibits the expression of MdCBF1 and MdCBF3 by directly binding to their promoters. It also interacts with MdICE1L, a homolog of AtICE1 in apple, and inhibits the binding of MdICE1L to the promoters of MdCBF1/3 and thus their expression. We showed that MdCAX3L-2, a CAX (Ca2+/H+ exchanger) family gene that negatively regulates plant cold tolerance, is also a direct target of MdbHLH4. MdbHLH4 reduced apple cold tolerance by promoting MdCAX3L-2 expression. Moreover, overexpression of either MdCAX3L-2 or MdbHLH4 promoted the cold-induced ubiquitination and degradation of MdICE1L. Overall, our results reveal that MdbHLH4 negatively regulates plant cold tolerance by inhibiting MdCBF1/3 expression and MdICE1L promoter-binding activity, as well as by promoting MdCAX3L-2 expression and cold-induced MdICE1L degradation. These findings provide insights into the mechanisms by which ICE1-interacting TFs regulate CBF expression and ICE1 function and thus plant cold tolerance.

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As the main fungal etiologic agent of apple (Malus domestica) replant disease (ARD), Fusarium solani seriously damages apple roots. ETHYLENE RESPONSE FACTORs (ERFs) play an important role in plant resistance to biotic stress. Here, we show that MdERF114 is expressed during F. solani infections and positively regulates the resistance of apple roots to F. solani. Yeast one-hybrid, dual-luciferase, electrophoretic mobility shift assays and determinations of lignin content indicated that MdERF114 directly binds the GCC-box of the MdPEROXIDASE63 (MdPRX63) promoter and activates its expression, resulting in lignin deposition in apple roots and increased resistance to F. solani. We identified a WRKY family transcription factor, MdWRKY75, that binds to the W-box of the MdERF114 promoter. Overexpression of MdWRKY75 enhanced resistance of apple roots to F. solani. MdMYB8 interacted with MdERF114 to enhance resistance to F. solani by promoting the binding of MdERF114 to the MdPRX63 promoter. In summary, our findings reveal that the MdWRKY75-MdERF114-MdMYB8-MdPRX63 module is required for apple resistance to F. solani and the application of this mechanism by Agrobacterium rhizogenes-mediated root transformation provides a promising strategy to prevent ARD.

SUMMARY Apple (Malus domestica) trees are vulnerable to freezing temperatures. Cold resistance in woody perennial plants can be improved through biotechnological approaches. However, genetic engineering requires a thorough understanding of the molecular mechanisms of the tree's response to cold. In this study, we demonstrated that the Mdm‐miR160–MdARF17–MdWRKY33 module is crucial for apple freezing tolerance. Mdm‐miR160 plays a negative role in apple freezing tolerance, whereas MdARF17, one of the targets of Mdm‐miR160, is a positive regulator of apple freezing tolerance. RNA sequencing analysis revealed that in apple, MdARF17 mediates the cold response by influencing the expression of cold‐responsive genes. EMSA and ChIP‐qPCR assays demonstrated that MdARF17 can bind to the promoter of MdWRKY33 and promotes its expression. Overexpression of MdWRKY33 enhanced the cold tolerance of the apple calli. In addition, we found that the Mdm‐miR160–MdARF17–MdWRKY33 module regulates cold tolerance in apple by regulating reactive oxygen species (ROS) scavenging, as revealed by (i) increased H2O2 levels and decreased peroxidase (POD) and catalase (CAT) activities in Mdm‐miR160e OE plants and MdARF17 RNAi plants and (ii) decreased H2O2 levels and increased POD and CAT activities in MdmARF17 OE plants and MdWRKY33 OE calli. Taken together, our study uncovered the molecular roles of the Mdm‐miR160–MdARF17–MdWRKY33 module in freezing tolerance in apple, thus providing support for breeding of cold‐tolerant apple cultivars.