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.

Regulation of Flowering Time by Improving Leaf Health Markers and Expansion by Salicylic Acid Treatment: A New Approach to Induce Flowering in Malus domestica.

In the external coincidence model, internal and external molecular signals, provided by the circadian clock and sunlight, respectively, are required to induce flowering. Salicylic acid (SA) applications during floral induction have multiple effects. In the current study, Malus × domestica plants were exposed to SA during the flower-induction stage to analyze the effect on various health markers and flowering. A total of 56 equal-sized Fuji/M9 trees that were about 7 years old were randomly divided into two groups. The first group (SA-treated) was sprayed with 4 mM SA solution, while the second group was sprayed with distilled water which served as control (CK). The SA applications increased various leaf pigments. Abiotic stress markers were increased in CK during the flower-induction stage. In the SA-treated group, non-enzymatic antioxidants increased, whereas in the control group, enzymatic antioxidants increased during the flower-induction stage. Histo-morphometric properties of leaves were significantly improved in the SA-treated group. The relative expression of the mRNA levels of MdMED80, -81, -3, and -41 were significantly increased in SA-treated leaves, leading to an early and increased flowering phenotype. Thus, SA increased leaf expansion and health-related marker levels, which lead to early induction of flowering in M. domestica. Overall, our work established a role for leaf health assessments in the regulation of flowering in M. domestica.

Zinc Finger-Homeodomain Genes: Evolution, Functional Differentiation, and Expression Profiling Under Flowering-Related Treatments and Abiotic Stresses in Plants.

Zinc finger-homeodomain (ZHD) proteins constitute a plant-specific transcription factor family that play important roles in plant growth, development, and stress responses. In this study, we investigated a total of 10, 17, and 31 ZHD gene members in the peach, Arabidopsis, and apple genome, respectively. The phylogenetic tree divided the identified ZHD genes into 4 subfamilies based on their domain organization, gene structure, and motif distribution with minor variations. The ZHD gene family members were unevenly distributed throughout in apple, peach, and Arabidopsis genomes. Segmental duplication was observed for 14 pairs of genes in apple. Transcript analysis found that ZHD genes mostly expressed in various tissues, particularly in leaves and flowers. Moreover, the transcript of most ZHD genes was significantly affected at different time points in response to various flowering-related exogenous hormones (sugar, gibberellin [GA], and 6-benzylaminopurine [6-BA]), signifying their possible role in the flowering induction in apple. Furthermore, the transcripts of CaZHD6, CaZHD7, CaZHD3, and CaZHD8 have induced in response to abiotic stresses including heat, drought, salt, and cold, indicating their possible involvement in response to abiotic stresses. Our research work systemically presents the different roles of ZHD genes. We believe that this study will provide a platform for future functional characterization of ZHD genes and to deeply unfold their roles in the regulation of flowering induction in plants.

Dynamic Cytosine DNA Methylation Patterns Associated with mRNA and siRNA Expression Profiles in Alternate Bearing Apple Trees.

Cytosine DNA methylation plays an important role in plants: it can mediate gene expression to affect plant growth and development. However, little is known about the potential involvement of cytosine DNA methylation in apple trees as well as in response to alternate bearing. Here, we performed whole-genome bisulfate sequencing to investigate genomic CG, CHG, and CHH methylation patterns, together with their global mRNA accumulation and small RNA expression in "Fuji" apple trees. Results showed that "Fuji" apple trees have a higher CHH methylation than Arabidopsis. Moreover, genomic methylation analysis revealed that CG and CHG methylation were robustly maintained at the early stage of flower induction. Additionally, differentially methylated regions (DMRs), including hypermethylated and hypomethylated DMRs, were also characterized in alternate bearing (AB) apple trees. Intriguingly, the DMRs were enriched in hormones, redox state, and starch and sucrose metabolism, which affected flowering. Further global gene expression evaluation based on methylome analysis revealed a negative correlation between gene body methylation and gene expression. Subsequent small RNA analyses showed that 24-nucleotide small interfering RNAs were activated and maintained in non-CG methylated apple trees. Our whole-genome DNA methylation analysis and RNA and small RNA expression profile construction provide valuable information for future studies.