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The low efficiency of somatic embryo induction, proliferation, germination, and conversion to plantlets is a major problem in walnut. In this study, we used the cotyledons of immature embryos of apomictic seeds in walnut plus trees 'Y17' as explants. The optimum medium and culture conditions of somatic embryo induction, proliferation, and germination were tested through comparative experiments. The results showed that the optimum formula for somatic embryo induction, proliferation, and germination medium was DKW medium with 1.0 mg L−1 6-BA, 2.0 mg L−1 KT, and 0.01 mg L−1 IBA, DKW medium with 30–40 g L−1 sucrose and 7–8 g L−1 agar, and DKW medium with 2.0 mg L−1 concentrations of GA3, respectively. Meanwhile, the optimum time of seedling training was 36 h, and the substrate ratio was perlite: peat soil: vermiculite (1: 2: 1) after plant transplanting. The inter simple sequence repeat marker banding patterns of the primary embryo, secondary embryo and regenerated plants were all identical to that of the mother plant and proved the genetic stability of regenerated plants. An excellent walnut tissue culture system from walnut somatic embryo to the whole plant was formed, which can accelerate the speed of walnut genetic improvement. Key message: An excellent walnut tissue culture system from walnut somatic embryo to the whole plant was formed, which can accelerate the speed of walnut genetic improvement. [ABSTRACT FROM AUTHOR]

Sugar content and cold tolerance are important apple (Malus × domestica) characteristics targeted by breeding programs. Here, a vacuolar membrane sucrose transporter, MdSWEET16, was cloned and characterized. A phylogenetic tree analysis found that MdSWEET16 was on the same polygenetic branch as Arabidopsis thaliana SWEET16 and SWEET17. MdSWEET16 was located on chromosome 2 and consists of six exons and five introns. The recombinant protein was obtained by prokaryotic induction, and the amino acid sequence and transmembrane domain were analyzed by bioinformatics. The encoded ~ 30-kDa protein has seven transmembrane domains and is localized on the tonoplasts of 'Orin' callus protoplasts. The expression of MdSWEET16 changed in response to sucrose and low temperature in 'Orin' calli. In addition, we also analyzed the expression level of MdSWEET16 during different fruit developmental stages using qRT-PCR. MdSWEET16 was highly expressed in young fruit, and its expression level during fruit development was significantly negatively correlated with sucrose content as assessed by a quantitative fluorescence analysis. Overexpression of MdSWEET16 in 'Orin' calli could reduce their sucrose content but increased their cold tolerance compared with MdSWEET16 RNA interference calli, which indicated that MdSWEET16 is involved in the sucrose transport and cold tolerance of apple. Key message: MdSWEET16's expression level was significantly correlated with the sucrose content and low temperature, and was induced by sucrose and low temperatures. Overexpression of MdSWEET16 increased 'Orin' calli cold tolerance. The prokaryotic expression of its recombinant protein can be used to further study the function of the MdSWEET16 protein in apple. [ABSTRACT FROM AUTHOR]

The WRKY transcription factors play key roles in plant growth and abiotic stress responses; however, the molecular mechanisms behind their involvement in anthocyanin biosynthesis are still unclear. In our study, we identified a Leu zipper motif and a WRKY domain in MdWRKY40 protein. Phylogenetic tree analysis showed that MdWRKY40, AtWRKY18 and AtWRKY40 were on the same evolutionary branch and were Group IIa WRKY proteins. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that MdWRKY40 could interact with itself to form homodimers. Overexpressing MdMYB111 in red-fleshed callus inhibited the expression of MdANS and decreased the anthocyanin content. EMSA assay showed that MdMYB111 and MdWRKY40 could bind the MRE and the W box, respectively, in the MdANS promoter. Overexpressing MdWRKY40 in red-fleshed callus did not affect the expression of MdANS or the anthocyanin content. However, overexpressing MdWRKY40 in callus overexpressing MdMYB111 weakened the inhibitory effect of MdMYB111 on anthocyanin biosynthesis. Knocking out the Leu zipper motif of MdWRKY40 (LLSMdWRKY40) prevented its self-interaction, and knocking out C-x5-C sequence of MdWRKY40 (LCSMdWRKY40) prevented it from binding to W box. It did not weaken the inhibitory effect of MdMYB111 on anthocyanin biosynthesis when overexpressing LCSMdWRKY40 or LLSMdWRKY40 in callus overexpressing MdMYB111. Thus, MdMYB111 and MdWRKY40 may play important roles in the anthocyanin biosynthetic pathway. Key message: MdWRKY40 interacts with itself to form homodimers by the Leu zipper motif at the N-terminal, and it binds two W boxes distantly separated in the MdANS promoter in the presence of C-x5-C sequence. MdMYB111 binds the MRE in the looped region induced by MdWRKY40. In addition, it weakens the inhibitory effect of MdMYB111 on expression of MdANS and anthocyanin biosynthesis when overexpressing MdWRKY40 in callus overexpressing MdMYB111. [ABSTRACT FROM AUTHOR]

Improving the cold resistance of plants is important, because their growth and development are negatively affected by cold stress. In this study, exogenous applications of methyl jasmonate could enhance the cold resistance of 'Orin' apple (Malus × domestica) calli by increasing the expression levels of the cold-signal response genes MdCIbHLH1, MdCBF1, MdCBF2 and MdCBF3. In addition, yeast two-hybrid and pull-down assays demonstrated that MdCIbHLH1 interacts with MdJAZ1/4 and MdMYC2 in vitro and in vivo. Protein degradation experiments demonstrated that the stability of the MdJAZ1/4 proteins were affected by the application of exogenous methyl jasmonate, which was followed by their degradation by the 26S proteasome. MdJAZ1/4 act as repressors, binding MdMYC2 in the jasmonate-signaling pathway. The overexpression of MdMYC2 in 'Orin' calli increased the expression levels of MdCIbHLH1, MdCBF1, MdCBF2 and MdCBF3, resulting in an increased freeze tolerance. Furthermore, the overexpression of MdJAZ1 or MdJAZ4 in transgenic red-fleshed apple calli weakened the promotive effect of MdMYC2 on cold tolerance. Yeast one-hybrid and chromatin immunoprecipitation-PCR analyses showed that MdMYC2 could bind the G-box element found in the MdCBF1 promoter. Thus, jasmonate may function as a critical upstream signal in the ICE-CBF/DREB1 pathway to positively regulate apple freeze tolerance. [ABSTRACT FROM AUTHOR]

As cold stress greatly affects plant growth and development, understanding the mechanisms underlying cold tolerance in plants is important. In this study, we analyzed the expression levels of apple (Malus domestica) MdbHLH33 and MdCBF1-5 by semi-quantitative PCR after exposure to 4 °C for different amounts of time and generated evolutionary trees for MdbHLH33 and the MdCBFs. Overexpressing MdbHLH33 pro-GUS in ‘Orin’ callus, indicated that transgenic callus had higher GUS activity and was more deeply stained at 4 °C than at 25 °C. Subcellular localization showed that MdbHLH33 was located in the nucleus. Overexpressing MdbHLH33 in ‘Orin’ callus increased the expression level of MdCBF2, MdCOR15A-1, and MdCOR15A-2, and resulted in increased cold tolerance. EMSA and Chip-PCR analysis showed that MdbHLH33 could bind the LTR cis-acting element found in the MdCBF2 promoter. Overexpressing MdCBF2 in ‘Orin’ callus indicated that MdCBF2 could also increase the expression level of MdCOR15A-1 and MdCOR15A-2 and improve cold tolerance; we also found that transgenic callus overexpressing MdCBF2 had reduced MdCBF1 and MdCBF5 expression and increased MdCBF3 and MdCBF4 expression. Overall, these results show that MdbHLH33 can regulate the expression of MdCBF2 and improve the cold tolerance of transgenic callus. [ABSTRACT FROM AUTHOR]

Exposure to high salt concentrations is one of the main stresses adversely affecting crop growth, development, and yield. In this study, a putative salt stress-responsive gene ( MdMYB4) encoding an R2R3-MYB transcription factor was cloned and characterized. This gene belongs to the SG2 subgroup, and its C-terminal contains a conserved SG2 motif. The relative MdMYB4 expression levels in Malus × domestica Borkh. 'Orin' calli cultured under various NaCl concentrations were analyzed. Saline conditions upregulated the expression of MdMYB4, and the effects of the encoded transcription factor on salt tolerance were determined in MdMYB4-overexpressing (OE-MYB4) 'Orin' calli. The OE-MYB4 calli grew significantly better than the wild-type calli under salt stress conditions. Additionally, an analysis of the cis-acting elements in the MdNHX1 and MdSOS1 promoters revealed MYB-binding sites. However, the electrophoretic mobility shift assay results indicated that MdMYB4 can bind only to the MdNHX1 promoter. Moreover, the MdNHX1 expression levels were higher in OE-MYB4 calli than in wild-type calli at 150, 200, and 250 mM NaCl. In contrast, there were no significant changes to the MdSOS1 expression levels in OE-MYB4 calli. In conclusion, MdMYB4 is an R2R3-MYB transcription factor that mediates salt tolerance by activating the MdNHX1 promoter. Our findings may provide a theoretical basis for enriching the genetic resources associated with stress tolerance and for improving stress resistance in apple cultivars. [ABSTRACT FROM AUTHOR]