Plant R2R3-MYB transcription factors (TFs) have been suggested to play crucial roles in the response to diverse abiotic and biotic stress factors but there is little molecular evidence of this role in soybean plants. In this work, we identified and functionally characterized an R2R3-MYB TF, namely, GsMYB15, from the wild soybean ED059. Protein and promoter sequence analysis indicated that GsMYB15 is a typical R2R3-MYB TF and contains multiple stress-related cis-elements in the promoter region. GsMYB15 is located in the nucleus and exhibits transcriptional activation activity. QPCR assays suggested that the expression of GsMYB15 could be induced by NaCl, insect attacks and defense-related hormones (MeJA and SA). Furthermore, GsMYB15 exhibited highest expression in pods compared to other tissues. Functional analysis of GsMYB15 demonstrated that overexpression of GsMYB15 could increase salt tolerance and enhance the resistance to H. armigera larvae in transgenic Arabidopsis plants. Moreover, overexpression of GsMYB15 also affected the expression levels of salt stress- and defense-related genes in the transgenic plants. Feeding with transgenic Arabidopsis plant leaves could significantly suppress the expression levels of immunity-related genes in H. armigera larvae. Overexpression of GsMYB15 also increased mesophyll cell levels in transgenic plants. Taken together, these results provide evidence that GsMYB15 is a positive regulator of salt stress tolerance and insect resistance in transformed Arabidopsis plants.

Fruit firmness is an important economical trait in sweet cherry (Prunus avium L.) where the change of this trait is related to cell wall degradation. Xyloglucan endotransglycosylase/hydrolase (XTH) and polygalacturonases (PGs) are critical cell-wall-modifying enzymes that occupy a crucial position in fruit ripening and softening. Herein, we identified 18 XTHs and 45 PGs designated PavXTH1-18 and PavPG1-45 based on their locations in the genome of sweet cherry. We provided a systematical overview of PavXTHs and PavPGs, including phylogenetic relationships, conserved motifs, and expression profiling of these genes. The results showed that PavXTH14, PavXTH15 and PavPG38 were most likely to participated in fruit softening owing to the substantial increment in expression during fruit development and ripening. Furthermore, the phytohormone ABA, MeJA, and ethephon significantly elevated the expression of PavPG38 and PavXTH15, and thus promoted fruit softening. Importantly, transient expression PavXTH14, PavXTH15 and PavPG38 in cherry fruits significantly reduced the fruit firmness, and the content of various cell wall components including hemicellulose and pectin significantly changed correspondingly in the transgenic fruit. Taken together, these results present an extensive analysis of XTHs and PGs in sweet cherry and provide potential targets for breeding softening-resistant sweet cherry cultivars via manipulating cell wall-associated genes.

Under drought stress, reactive oxygen species (ROS) overaccumulate as a secondary stress that impairs plant performance and thus severely reduces crop yields. The mitigation of ROS levels under drought stress is therefore crucial for drought tolerance. MicroRNAs (miRNAs) are critical regulators of plant development and stress responses. However, the complex molecular regulatory mechanism by which they function during drought stress, especially in drought-triggered ROS scavenging, is not fully understood. Here, we report a newly identified drought-responsive miRNA, miR164g, in the wild apple species Malus sieversii and elucidate its role in apple drought tolerance. Our results showed that expression of miR164g is significantly inhibited under drought stress and it can specifically cleave transcripts of the transcription factor MsNAC022 in M. sieversii. The heterologous accumulation of miR164g in Arabidopsis thaliana results in enhanced sensitivity to drought stress, while overexpression of MsNAC022 in Arabidopsis and the cultivated apple line ‘GL-3’ (Malus domestica Borkh.) lead to enhanced tolerance to drought stress by raising the ROS scavenging enzymes activity and related genes expression levels, particularly PEROXIDASE (MsPOD). Furthermore, we showed that expression of MsPOD is activated by MsNAC022 in transient assays. Interestingly, Part1 (P1) region is the key region for the positive regulation of MsPOD promoter by MsNAC022, and the different POD expression patterns in M. sieversii and M. domestica is attributed to the specific fragments inserted in P1 region of M. sieversii. Our findings reveal the function of the miR164g-MsNAC022 module in mediating the drought response of M. sieversii and lay a foundation for breeding drought-tolerant apple cultivars.

Plant-specific SQUAMOSA promoter-binding protein-like (SPL) transcription factors play important regulatory roles during plant growth and development, fruit ripening, inflorescence branching, and biotic and abiotic stresses. However, there have been no identification or systematic studies of the SPL gene family in the sweet cherry. In this study, 12 SPL genes were identified in the sweet cherry reference genome, which were distributed over 6 chromosomes and classified into six groups according to phylogenetic relationships with other SPL gene families. Nine PavSPLs were highly expressed at green fruit stages and dramatically decreased at the onset of fruit ripening, which implied that they were important regulators during fruit development and ripening. The expression patterns of PavSPL genes under ABA, GA, and MeJA treatments showed that the PavSPLs were involved in the process of fruit ripening. A subcellular localization experiment proved that PavSPL4 and PavSPL7 proteins were localized in the nucleus. The genome-wide identification of the SPL gene family provided new insights while establishing an important foundation for sweet cherry studies.

B-BOX proteins are zinc finger transcription factors that play important roles in plant growth, development, and abiotic stress responses. In this study, we identified 15 PavBBX genes in the genome database of sweet cherry. We systematically analyzed the gene structures, clustering characteristics, and expression patterns of these genes during fruit development and in response to light and various hormones. The PavBBX genes were divided into five subgroups. The promoter regions of the PavBBX genes contain cis-acting elements related to plant development, hormones, and stress. qRT-PCR revealed five upregulated and eight downregulated PavBBX genes during fruit development. In addition, PavBBX6, PavBBX9, and PavBBX11 were upregulated in response to light induction. We also found that ABA, BR, and GA₃ contents significantly increased in response to light induction. Furthermore, the expression of several PavBBX genes was highly correlated with the expression of anthocyanin biosynthesis genes, light-responsive genes, and genes that function in multiple hormone signaling pathways. Some PavBBX genes were strongly induced by ABA, GA, and BR treatment. Notably, PavBBX6 and PavBBX9 responded to all three hormones. Taken together, BBX proteins likely play major roles in regulating anthocyanin biosynthesis in sweet cherry fruit by integrating light, ABA, GA, and BR signaling pathways.

Drought is the main environmental factor that limits the yield and quality of apples (Malus × domestica) grown in arid and semi-arid regions. Nuclear factor Ys (NF-Ys) are important transcription factors involved in the regulation of plant growth, development, and various stress responses. However, the function of NF-Y genes is poorly understood in apples. Here, we identified 43 NF-Y genes in the genome of apples and conducted an initial functional characterization of the apple NF-Y. Expression analysis of NF-Y members in M. sieversii revealed that a large number of NF-Ys were highly expressed in the roots compared with the leaves, and a large proportion of NF-Y genes responded to drought treatment. Furthermore, heterologous expression of MsNF-YB21, which was significantly upregulated by drought, led to a longer root length and, thus, conferred improved osmotic and salt tolerance in Arabidopsis. Moreover, the physiological analysis of MsNF-YB21 overexpression revealed enhanced antioxidant systems, including antioxidant enzymes and compatible solutes. In addition, genes encoding catalase (AtCAT2, AtCAT3), superoxide dismutase (AtFSD1, AtFSD3, AtCSD1), and peroxidase (AtPER12, AtPER42, AtPER47, AtPER51) showed upregulated expression in the MsNF-YB21 overexpression lines. These results for the MsNF-Y gene family provide useful information for future studies on NF-Ys in apples, and the functional analysis of MsNF-YB21 supports it as a potential target in the improvement of apple drought tolerance via biotechnological strategies.