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Summary: RAD5B belongs to the Rad5/16‐like group of the SNF2 family, which often functions in chromatin remodelling. However, whether RAD5B is involved in chromatin remodelling, histone modification, and drought stress tolerance is largely unclear. We identified a drought‐inducible chromatin remodeler, MdRAD5B, which positively regulates apple drought tolerance. Transposase‐accessible chromatin with high‐throughput sequencing (ATAC‐seq) analysis showed that MdRAD5B affects the expression of 466 drought‐responsive genes through its chromatin remodelling function in response to drought stress. In addition, MdRAD5B interacts with and degrades MdLHP1, a crucial regulator of histone H3 trimethylation at K27 (H3K27me3), through the ubiquitin‐independent 20S proteasome. Chromatin immunoprecipitation‐sequencing (ChIP‐seq) analysis revealed that MdRAD5B modulates the H3K27me3 deposition of 615 genes in response to drought stress. Genetic interaction analysis showed that MdRAD5B mediates the H3K27me3 deposition of drought‐responsive genes through MdLHP1, which causes their expression changes under drought stress. Our results unravelled a dual function of MdRAD5B in gene expression modulation in apple in response to drought, that is, via the regulation of chromatin remodelling and H3K27me3. [ABSTRACT FROM AUTHOR]

Summary: Although the N6‐methyladenosine (m6A) modification is the most prevalent RNA modification in eukaryotes, the global m6A modification landscape and its molecular regulatory mechanism in response to drought stress remain unclear.Transcriptome‐wide m6A methylome profiling revealed that m6A 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 m6A methyltransferase complex, MdMTA. An in vitro methyl transfer assay, dot blot, LC‐MS/MS and m6A‐sequencing (m6A‐seq) suggested that MdMTA is an m6A writer and essential for m6A mRNA modification.Further studies revealed that MdMTA is required for apple drought tolerance. m6A‐seq and RNA‐seq analyses under drought conditions showed that MdMTA mediates m6A modification and transcripts of mRNAs involved in oxidative stress and lignin deposition. Moreover, m6A 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 m6A 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. [ABSTRACT FROM AUTHOR]

SUMMARY: Drought significantly limits apple fruit production and quality. Decoding the key genes involved in drought stress tolerance is important for breeding varieties with improved drought resistance. Here, we identified GRETCHEN HAGEN3.6 (GH3.6), an indole‐3‐acetic acid (IAA) conjugating enzyme, to be a negative regulator of water‐deficit stress tolerance in apple. Overexpressing MdGH3.6 reduced IAA content, adventitious root number, root length and water‐deficit stress tolerance, whereas knocking down MdGH3.6 and its close paralogs increased IAA content, adventitious root number, root length and water‐deficit stress tolerance. Moreover, MdGH3.6 negatively regulated the expression of wax biosynthetic genes under water‐deficit stress and thus negatively regulated cuticular wax content. Additionally, MdGH3.6 negatively regulated reactive oxygen species scavengers, including antioxidant enzymes and metabolites involved in the phenylpropanoid and flavonoid pathway in response to water‐deficit stress. Further study revealed that the homolog of transcription factor AtMYB94, rather than AtMYB96, could bind to the MdGH3.6 promoter and negatively regulated its expression under water‐deficit stress conditions in apple. Overall, our results identify a candidate gene for the improvement of drought resistance in fruit trees. Significance Statement: We characterized MdGH3.6 in the response of apple to water‐deficit stress. We found that MdGH3.6 was a direct target of MdMYB94 and a negative regulator of water‐deficit stress tolerance in apple. The negative regulation of water‐deficit stress tolerance may stem from the combined roles of MdGH3.6 in the negative regulation of ROS detoxification and cuticular wax. Overall, our results identify a candidate gene for the improvement of drought resistance in fruit trees. [ABSTRACT FROM AUTHOR]

Drought stress is a significant environmental factor limiting crop growth worldwide. Malus prunifolia is an important apple species endemic to China and is used for apple cultivars and rootstocks with great drought tolerance. N6-methyladenosine (m6A) is a common epigenetic modification on messenger RNAs (mRNAs) in eukaryotes which is critical for various biological processes. However, there are no reports on m6A methylation in apple response to drought stress. Here, we assessed the m6A landscape of M. prunifolia seedlings in response to drought and analyzed the association between m6A modification and transcript expression. In total, we found 19,783 and 19,609 significant m6A peaks in the control and drought treatment groups, respectively, and discovered a UGUAH (H: A/U/C) motif. In M. prunifolia, under both control and drought conditions, peaks were highly enriched in the 3′ untranslated region (UTR) and coding sequence (CDS). Among 4204 significant differential m6A peaks in drought-treated M. prunifolia compared to control-treated M. prunifolia, 4158 genes with m6A modification were identified. Interestingly, a large number of hypermethylated peaks (4069) were stimulated by drought treatment compared to hypomethylation. Among the hypermethylated peak-related genes, 972 and 1238 differentially expressed genes (DEGs) were up- and down-regulated in response to drought, respectively. Gene ontology (GO) analyses of differential m6A-modified genes revealed that GO slims related to RNA processing, epigenetic regulation, and stress tolerance were significantly enriched. The m6A modification landscape depicted in this study sheds light on the epigenetic regulation of M. prunifolia in response to drought stress and indicates new directions for the breeding of drought-tolerant apple trees. [ABSTRACT FROM AUTHOR]

Although the N 6 -methyladenosine (m 6 A) modification is the most prevalent RNA modification in eukaryotes, the global m 6 A modification landscape and its molecular regulatory mechanism in response to drought stress remain unclear. Transcriptome-wide m 6 A methylome profiling revealed that m 6 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 m 6 A methyltransferase complex, MdMTA. An in vitro methyl transfer assay, dot blot, LC-MS/MS and m 6 A-sequencing (m 6 A-seq) suggested that MdMTA is an m 6 A writer and essential for m 6 A mRNA modification. Further studies revealed that MdMTA is required for apple drought tolerance. m 6 A-seq and RNA-seq analyses under drought conditions showed that MdMTA mediates m 6 A modification and transcripts of mRNAs involved in oxidative stress and lignin deposition. Moreover, m 6 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 m 6 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., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Drought stress tolerance is a complex trait regulated by multiple factors. Here, we demonstrate that the miRNA160-Auxin Response Factor 17 (ARF17)-HYPONASTIC LEAVES 1 module is crucial for apple (Malus domestica) drought tolerance. Using stable transgenic plants, we found that drought tolerance was improved by higher levels of Mdm-miR160 or MdHYL1 and by decreased levels of MdARF17, whereas reductions in MdHYL1 or increases in MdARF17 led to greater drought sensitivity. Further study revealed that modulation of drought tolerance was achieved through regulation of drought-responsive miRNA levels by MdARF17 and MdHYL1; MdARF17 interacted with MdHYL1 and bound to the promoter of MdHYL1. Genetic analysis further suggested that MdHYL1 is a direct downstream target of MdARF17. Importantly, MdARF17 and MdHYL1 regulated the abundance of Mdm-miR160. In addition, the Mdm-miR160-MdARF17-MdHYL1 module regulated adventitious root development. We also found that Mdm-miR160 can move from the scion to the rootstock in apple and tomato (Solanum lycopersicum), thereby improving root development and drought tolerance of the rootstock. Our study revealed the mechanisms by which the positive feedback loop of Mdm-miR160-MdARF17-MdHYL1 influences apple drought tolerance., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Water deficit is one of the main challenges for apple (Malus × domestica) growth and productivity. Breeding drought-tolerant cultivars depends on a thorough understanding of the drought responses of apple trees. Here, we identified the zinc-finger protein B-BOX 7/CONSTANS-LIKE 9 (MdBBX7/MdCOL9), which plays a positive role in apple drought tolerance. The overexpression of MdBBX7 enhanced drought tolerance, whereas knocking down MdBBX7 expression reduced it. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis identified one cis-element of MdBBX7, CCTTG, as well as its known binding motif, the T/G box. ChIP-seq and RNA-seq identified 1,197 direct targets of MdBBX7, including ETHYLENE RESPONSE FACTOR (ERF1), EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15), and GOLDEN2-LIKE 1 (GLK1) and these were further verified by ChIP-qPCR and electronic mobility shift assays. Yeast two-hybrid screen identified an interacting protein of MdBBX7, RING-type E3 ligase MYB30-INTERACTING E3 LIGASE 1 (MIEL1). Further examination revealed that MdMIEL1 could mediate the ubiquitination and degradation of MdBBX7 by the 26S proteasome pathway. Genetic interaction analysis suggested that MdMIEL1 acts as an upstream factor of MdBBX7. In addition, MdMIEL1 was a negative regulator of the apple drought stress response. Taken together, our results illustrate the molecular mechanisms by which the MdMIEL1-MdBBX7 module influences the response of apple to drought stress., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Drought stress is a significant environmental factor limiting crop growth worldwide. Malus prunifolia is an important apple species endemic to China and is used for apple cultivars and rootstocks with great drought tolerance. N 6 -methyladenosine (m 6 A) is a common epigenetic modification on messenger RNAs (mRNAs) in eukaryotes which is critical for various biological processes. However, there are no reports on m 6 A methylation in apple response to drought stress. Here, we assessed the m 6 A landscape of M. prunifolia seedlings in response to drought and analyzed the association between m 6 A modification and transcript expression. In total, we found 19,783 and 19,609 significant m 6 A peaks in the control and drought treatment groups, respectively, and discovered a UGUAH (H: A/U/C) motif. In M. prunifolia , under both control and drought conditions, peaks were highly enriched in the 3' untranslated region (UTR) and coding sequence (CDS). Among 4204 significant differential m 6 A peaks in drought-treated M. prunifolia compared to control-treated M. prunifolia , 4158 genes with m 6 A modification were identified. Interestingly, a large number of hypermethylated peaks (4069) were stimulated by drought treatment compared to hypomethylation. Among the hypermethylated peak-related genes, 972 and 1238 differentially expressed genes (DEGs) were up- and down-regulated in response to drought, respectively. Gene ontology (GO) analyses of differential m 6 A-modified genes revealed that GO slims related to RNA processing, epigenetic regulation, and stress tolerance were significantly enriched. The m 6 A modification landscape depicted in this study sheds light on the epigenetic regulation of M. prunifolia in response to drought stress and indicates new directions for the breeding of drought-tolerant apple trees.