Genome-Wide Identification of the DOF Gene Family in Kiwifruit (Actinidia chinensis) and Functional Validation of AcDOF22 in Response to Drought Stress.

DNA-binding one zinc finger (DOF) transcription factors are crucial plant-specific regulators involved in growth, development, signal transduction, and abiotic stress response generation. However, the genome-wide identification and characterization of AcDOF genes and their regulatory elements in kiwifruit (Actinidia chinensis) has not been thoroughly investigated. In this study, we screened the kiwifruit genome database and identified 42 AcDOF genes (AcDOF1 to AcDOF42). Phylogenetic analysis facilitated the categorization of these genes into five subfamilies (DOF-a, DOF-b, DOF-c, DOF-d, and DOF-e). We further analyzed the motifs, conserved domains, gene structures, and collinearity of the AcDOFgene family. Gene ontology (GO) enrichment analysis indicated significant enrichment in the "flower development" term and the "response to abiotic stress" category. Promoter prediction analysis revealed numerous cis-regulatory elements related to responses to light, hormones, and low-temperature and drought stress in AcDOF promoters. RNA-seq expression profiles demonstrated the tissue-specific expression of AcDOF genes. Quantitative real-time PCR results showed that six selected genes (AcDOF04, AcDOF09, AcDOF11, AcDOF13, AcDOF21, and AcDOF22) were differentially induced by abscisic acid (ABA), methyl jasmonate (MeJA), and cold, salt, and drought stresses, with AcDOF22 specifically expressed at high levels in drought-tolerant cultivars. Further experiments indicated that transient AcDOF22 overexpression in kiwifruit leaf disks reduced water loss and chlorophyll degradation. Additionally, AcDOF22 was localized to the nucleus and exhibited transcriptional activation, enhancing drought resistance by activating the downstream drought marker gene AcDREB2A. These findings lay the foundation for elucidating the molecular mechanisms of drought resistance in kiwifruit and offer new insights into drought-resistant breeding.

MdMTA-mediated m6 A modification enhances drought tolerance by promoting mRNA stability and translation efficiency of genes involved in lignin deposition and oxidative stress.

Although the N6 -methyladenosine (m6 A) modification is the most prevalent RNA modification in eukaryotes, the global m6 A modification landscape and its molecular regulatory mechanism in response to drought stress remain unclear. Transcriptome-wide m6 A methylome profiling revealed that m6 A is mainly enriched in the coding sequence and 3' untranslated region in response to drought stress in apple, by recognizing the plant-specific sequence motif UGUAH (H=A, U or C). We identified a catalytically active component of the m6 A methyltransferase complex, MdMTA. An in vitro methyl transfer assay, dot blot, LC-MS/MS and m6 A-sequencing (m6 A-seq) suggested that MdMTA is an m6 A writer and essential for m6 A mRNA modification. Further studies revealed that MdMTA is required for apple drought tolerance. m6 A-seq and RNA-seq analyses under drought conditions showed that MdMTA mediates m6 A modification and transcripts of mRNAs involved in oxidative stress and lignin deposition. Moreover, m6 A modification promotes mRNA stability and the translation efficiency of these genes in response to drought stress. Consistently, MdMTA enhances lignin deposition and scavenging of reactive oxygen species under drought conditions. Our results reveal the global involvement of m6 A modification in the drought response of perennial apple trees and illustrate its molecular mechanisms, thereby providing candidate genes for the breeding of stress-tolerant apple cultivars.

A multifaceted module of BRI1 ETHYLMETHANE SULFONATE SUPRESSOR1 (BES1)-MYB88 in growth and stress tolerance of apple.

The R2R3 transcription factor MdMYB88 has previously been reported to function in biotic and abiotic stress responses. Here, we identify BRI1 ETHYLMETHANE SULFONATE SUPRESSOR1 (MdBES1), a vital component of brassinosteroid (BR) signaling in apple (Malus × domestica) that directly binds to the MdMYB88 promoter, regulating the expression of MdMYB88 in a dynamic and multifaceted mode. MdBES1 positively regulated expression of MdMYB88 under cold stress and pathogen attack, but negatively regulated its expression under control and drought conditions. Consistently, MdBES1 was a positive regulator for cold tolerance and disease resistance in apple, but a negative regulator for drought tolerance. In addition, MdMYB88 participated in BR biosynthesis by directly regulating the BR biosynthetic genes DE ETIOLATED 2 (MdDET2), DWARF 4 (MdDWF4), and BRASSINOSTEROID 6 OXIDASE 2 (MdBR6OX2). Applying exogenous BR partially rescued the erect leaf and dwarf phenotypes, as well as defects in stress tolerance in MdMYB88/124 RNAi plants. Moreover, knockdown of MdMYB88 in MdBES1 overexpression (OE) plants decreased resistance to a pathogen and C-REPEAT BINDING FACTOR1 expression, whereas overexpressing MdMYB88 in MdBES1 OE plants increased expression of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 （MdSPL3） and BR biosynthetic genes, suggesting that MdMYB88 contributes to MdBES1 function during BR biosynthesis and the stress response. Taken together, our results reveal multifaceted regulation of MdBES1 on MdMYB88 in BR biosynthesis and stress tolerance.

Cold shock protein 3 plays a negative role in apple drought tolerance by regulating oxidative stress response.

As RNA chaperones, cold shock proteins (CSPs) are essential for cold adaptation. Although the functions of CSPs in cold response have been demonstrated in several species, the roles of CSPs in response to drought are largely unknown. Here, we demonstrated that MdCSP3, a downstream target gene of MdMYB88 and MdMYB124, contributes to drought tolerance in apple (Malus × domestica). MdCSP3 responds to various abiotic stresses, including drought, cold, heat, and salt stress. Compared with non-transgenic apple GL-3, the MdCSP3 overexpressing plants exhibit significantly lower drought resistance and a reduced capacity for ROS scavenging by the regulation of antioxidant enzymes SOD, CAT, and POD. Additionally, RNA-seq data shows that MdCSP3 regulates expression of genes involved in oxidative stress response. Taken together, our results demonstrate the functions of MdCSP3 in apple drought tolerance, and this finding provides a new direction for breeding of drought resistant apple.

FISH-based mitotic and meiotic diakinesis karyotypes of Morus notabilis reveal a chromosomal fusion-fission cycle between mitotic and meiotic phases.

Mulberry (Morus spp.), in family Moraceae, is a plant with important economic value. Many polyploid levels of mulberry have been determined. In the present study, the fluorescence in situ hybridization (FISH) technique was applied in Morus notabilis, using four single-copy sequences, telomere repeats, and 5S and 25S rDNAs as probes. All the mitotic chromosomes were clearly identified and grouped into seven pairs of homologous chromosomes. Three dot chromosome pairs were distinguished by the FISH patterns of the 25S rDNA probe and a simple sequence repeat (SSR2524). According to the FISH signals, chromosome length and morphology, detailed meiotic diakinesis karyotype was constructed. Interestingly, only six bivalent chromosomes were observed in diakinesis cells. The 25S rDNA probe was used to illustrate chromosome alterations. The results indicated that mitotic chromosomes 5 and 7 fused into diakinesis chromosome 5 during the meiotic phase. In mitotic cells, the fused chromosome 5 broke into chromosomes 5 and 7. A chromosomal fusion-fission cycle between the meiotic and mitotic phases in the same individual is reported here for the first time. This finding will contribute to the understanding of karyotype evolution in plants.