The positive feedback regulatory loop of miR160-Auxin Response Factor 17-HYPONASTIC LEAVES 1 mediates drought tolerance in apple trees.

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.

A genome-wide association study provides insights into fatty acid synthesis and metabolism in Malus fruits.

As a precursor of aromatic compounds, fatty acids play important roles in apple fruit quality; however, the genetic and molecular basis underlying fatty acid synthesis and metabolism is largely unknown. In this study, we conducted a genome-wide association study (GWAS) of seven fatty acids using genomic data of 149 Malus accessions and identified 232 significant signals (-log10P>5) associated with 99 genes from GWAS of four fatty acids across 2 years. Among these, a significant GWAS signal associated with linoleic acid was identified in the transcriptional regulator SUPERMAN-like (SUP) MD13G1209600 at chromosome 13 of M. × domestica. Transient overexpression of MdSUP increased the contents of linoleic and linolenic acids and of three aromatic components in the fruit. Our study provides genetic and molecular information for improving the flavor and nutritional value of apple.

[Winter pruning boosts growth and yield of Lonicera japonica by regulating plant hormone content].

Lonicera japonica is a ubiquitous medicinal species in China.Winter pruning has long been used to improve its quality and yield, but the mechanism is rarely studied.Therefore, in this study, the growth phenotypes of L.japonica processed with different pruning methods were observed and the yield-and quality-boosting mechanism of pruning was analyzed.Specifically, the young shoots of the three-year old L.japonica were cut to different degrees(heavy pruning, mild pruning, and no pruning, respectively) in winter in 2020 and 2021, respectively, and the growth phenotypes, hormone content, and gene expression of the lateral buds at the sprouting stage and young shoots at the anthesis stage in the next year were analyzed.The result showed that the length, flower bud number, internode length, and node number of young shoots in the next year were in the order of heavy pruning>mild pruning>no pruning.The content of auxin and zeatin in apical buds of young shoots at the anthesis stage was the highest in the heavy pruning group, followed by the mild pruning group, and coming in the third was the no pruning group.The content of auxin and zeatin in lateral buds at the sprouting stage was in the order of no pruning>mild pruning>heavy pruning.Transcriptome analysis of the lateral buds at sprouting stage yielded the differentially expressed genes related to auxin and cytokinin, such as Lj1A1163T36, Lj3A719T115, Lj7C657T7, Lj9C505T15, and Lj9A505T70.In conclusion, the growth phenotypes of young shoots of L.japonica processed with different pruning methods in winter were related to the difference in hormone content in the apical buds.Therefore, winter pruning influenced the content of auxin and cytokinin in new shoots of L.japonica and further regulated the expression of hormone-related genes, thereby promoting shoot growth and increasing the yield of L.japonica.

Insights into the effect of human civilization on Malus evolution and domestication.

The evolutionary history of the Malus genus has not been well studied. In the current study, we presented genetic evidence on the origin of the Malus genus based on genome sequencing of 297 Malus accessions, revealing the genetic relationship between wild species and cultivated apples. Our results demonstrated that North American and East Asian wild species are closer to the outgroup (pear) than Central Asian species, and hybrid species including natural (separated before the Pleistocene, about 2.5 Mya) and artificial hybrids (including ornamental trees and rootstocks) are between East and Central Asian wild species. Introgressions from M. sylvestris in cultivated apples appeared to be more extensive than those from M. sieversii, whose genetic background flowed westward across Eurasia and eastward to wild species including M. prunifolia, M. × asiatica, M. × micromalus, and M. × robust. Our results suggested that the loss of ancestral gene flow from M. sieversii in cultivated apples accompanied the movement of European traders around the world since the Age of Discovery. Natural SNP variations showed that cultivated apples had higher nucleotide diversity than wild species and more unique SNPs than other apple groups. An apple ERECTA-like gene that underwent selection during domestication on 15th chromosome was identified as a likely major determinant of fruit length and diameter, and an NB-ARC domain-containing gene was found to strongly affect anthocyanin accumulation using a genome-wide association approach. Our results provide new insights into the origin and domestication of apples and will be useful in new breeding programmes and efforts to increase fruit crop productivity.

Abscisic acid homeostasis is mediated by feedback regulation of MdMYB88 and MdMYB124.

The phytohormone abscisic acid (ABA) is involved in various plant processes. In response to drought stress, plants quickly accumulate ABA, but the regulatory mechanism of ABA accumulation is largely unknown, especially in woody plants. In this study, we report that MdMYB88 and MdMYB124 are myeloblastosis (MYB) transcription factors critical for ABA accumulation in apple trees (Malus x domestica) following drought, and this regulation is negatively controlled by ABA. MdMYB88 and MdMYB124 positively regulate leaf water transpiration, photosynthetic capacity, and stress endurance in apple trees under drought conditions. MdMYB88 and MdMYB124 regulate the expression of biosynthetic and catabolic genes of ABA, as well as drought- and ABA- responsive genes. MdMYB88 associates with promoter regions of the ABA biosynthetic gene 9-cis-epoxycarotenoid dioxygenase 3 (NCED3). Finally, expression of MdMYB88 and MdMYB124 is repressed by ABA. Our results identify a feedback regulation of MdMYB88 and MdMYB124 in modulating ABA homeostasis in apple trees.

Integrative Analyses of Widely Targeted Metabolic Profiling and Transcriptome Data Reveals Molecular Insight into Metabolomic Variations during Apple (Malus domestica) Fruit Development and Ripening.

The apple is a favorite fruit for human diet and is one of the most important commercial fruit crops around the world. Investigating metabolic variations during fruit development can provide a better understanding on the formation of fruit quality. The present study applied a widely targeted LC-MS-based metabolomics approach with large-scale detection, identification and quantification to investigate the widespread metabolic changes during "Pinova" apple development and ripening. A total of 462 primary and secondary metabolites were simultaneously detected, and their changes along with the four fruit-development stages were further investigated. The results indicated that most of the sugars presented increasing accumulation levels while organic acid, including Tricarboxylic acid cycle (TCA) intermediates, showed a distinct decreasing trend across the four fruit-development stages. A total of 207 secondary metabolites consisted of 104 flavonoids and 103 other secondary metabolites. Many flavonoids maintained relatively high levels in the early fruit stage and then rapidly decreased their levels at the following developmental stages. Further correlation analyses of each metabolite-metabolite pair highlighted the cross talk between the primary and secondary metabolisms across fruit development and ripening, indicating the significant negative correlations between sugars and secondary metabolites. Moreover, transcriptome analysis provided the molecular basis for metabolic variations during fruit development. The results showed that most differentially expressed genes (DEGs) involved in the TCA cycle were upregulated from the early fruit stage to the preripening stage. The extensive downregulation of controlling genes involved in the flavonoid pathway is probably responsible for the rapid decrease of flavonoid content at the early fruit stage. These data provide a global view of the apple metabolome and a comprehensive analysis on metabolomic variations during fruit development, providing a broader and better understanding on the molecular and metabolic basis of important fruit quality traits in commercial apples.

MdMYB88 and MdMYB124 Enhance Drought Tolerance by Modulating Root Vessels and Cell Walls in Apple.

Water deficit is one of the main limiting factors in apple (Malus × domestica Borkh.) cultivation. Root architecture plays an important role in the drought tolerance of plants; however, research efforts to improve drought tolerance of apple trees have focused on aboveground targets. Due to the difficulties associated with visualization and data analysis, there is currently a poor understanding of the genetic players and molecular mechanisms involved in the root architecture of apple trees under drought conditions. We previously observed that MdMYB88 and its paralog MdMYB124 regulate apple tree root morphology. In this study, we found that MdMYB88 and MdMYB124 play important roles in maintaining root hydraulic conductivity under long-term drought conditions and therefore contribute toward adaptive drought tolerance. Further investigation revealed that MdMYB88 and MdMYB124 regulate root xylem development by directly binding MdVND6 and MdMYB46 promoters and thus influence expression of their target genes under drought conditions. In addition, MdMYB88 and MdMYB124 were shown to regulate the deposition of cellulose and lignin root cell walls in response to drought. Taken together, our results provide novel insights into the importance of MdMYB88 and MdMYB124 in root architecture, root xylem development, and secondary cell wall deposition in response to drought in apple trees.

A 13-LOX participates in the biosynthesis of JAs and is related to the accumulation of baicalein and wogonin in Scutellaria baicalensis.

Although baicalein and wogonin contents in Scutellaria baicalensis, a traditional Chinese herb, are known to be regulated by jasmonic acid, the exact mechanism by which jasmonic acid regulates the accumulation of baicalein and wogonin remains unclear. In this study, we discovered SbLOX3, a gene encoding 13-lipoxygenase from the roots of S. baicalensis, which plays an important role in the biosynthesis of jasmonic acid. The contents of methyl jasmonate, baicalin, wogonin, and three metabolic intermediates of methyl jasmonate, 13-HPOT, OPDA, and OPC-8, were downregulated in the hair roots of the SbLOX3 RNAi lines. We confirmed that SbLOX3 was induced by drought stress simulated by PEG and Fusarium oxysporum, which subsequently led to changes in the content of MeJA, baicalin, and wogonin. Taken together, our results indicate that a 13-LOX is involved in the biosynthesis of jasmonic acid, and regulates the accumulation of baicalein and wogonin in S. baicalensis roots.

MeJA regulates the accumulation of baicalein and other 4'-hydroxyflavones during the hollowed root development in Scutellaria baicalensis.

The dried roots of Scutellaria baicalensis are important traditional Chinese medicine used to treat liver and lung inflammation. An anomalous structure, hollowed root, was discovered in perennial cultivated Scutellaria baicalensis. The presence of the hollow may change the contents of bioactive metabolites, such as baicalein, and other 4'-hydroxyflavones in Scutellaria baicalensis roots, but the relationship between the hollowed root and bioactive metabolite contents is poorly understood. In this study, we identified the anatomical structure of the hollowed root and detected differentially accumulating flavonoid metabolites and enzymes related to 4'-hydroxyflavone biosynthesis in 3-year-old roots with a hollow. We confirmed that methyl jasmonate (MeJA) induced the accumulation of 4'-hydroxyflavones and the expression of enzymes related to 4'-hydroxyflavone biosynthesis in hydroponically cultured Scutellaria baicalensis roots. The development of the hollowed root were divided into 4 stages. The 4'-hydroxyflavone contents and expression of enzymes related to 4'-hydroxyflavone biosynthesis increased synchronously with the content of MeJA during the development of hollowed root. Pathogen and programed-cell-death related genes were induced during hollowed root development. Taken together, our results provide novel insight into the importance of MeJA in the development of hollowed root and the accumulation of 4'-hydroxyflavones in Scutellaria baicalensis roots. Our results suggest that a pathogen and senescence are the two major causes for the development of hollowed root in Scutellaria baicalensis roots.

Regulation of phenylpropanoid biosynthesis by MdMYB88 and MdMYB124 contributes to pathogen and drought resistance in apple.

MdMYB88 and MdMYB124 have been demonstrated to be responsible for lignin accumulation in apple under drought stress. In this study, using a metabolomic approach, we identified differentially accumulated phenylpropanoid and flavonoid metabolites in MdMYB88/124 transgenic RNAi plants under control and long-term drought stress conditions in apple roots. We confirmed the regulation of phenylalanine by MdMYB88 and MdMYB124 via UPLC-MS in apple roots under both control and drought conditions. Using Electrophoretic Mobility Shift Assay (EMSA) and ChIP-quantitative PCR (qPCR) analyses, we found that MdMYB88 positively regulates the MdCM2 gene, which is responsible for phenylalanine biosynthesis, through binding to its promoter region. Under long-term drought conditions, MdMYB88/124 RNAi plants consistently accumulated increased amounts of H2O2 and MDA, while MdMYB88 and MdMYB124 overexpression plants accumulated decreased amounts of H2O2 and MDA. We also examined the accumulation of metabolites in the phenylpropanoid biosynthesis pathway in the leaves of MdMYB88 and MdMYB124 transgenic apple plants after long-term drought stress. We found that metabolites responsible for plant defense, including phenylpropanoids and flavonoids, accumulated less in the RNAi plants but more in the overexpression plants under both control and drought conditions. We further demonstrated that MdMYB88/124 RNAi plants were more sensitive to Alternaria alternata f. sp. mali and Valsa mali, two pathogens that currently severely threaten apple production. In contrast, MdMYB88 and MdMYB124 overexpression plants were more tolerant to these pathogens. The cumulative results of this study provided evidence for secondary metabolite regulation by MdMYB88 and MdMYB124, further explained the molecular roles of MdMYB88 and MdMYB124 in drought resistance, and provided information concerning molecular aspects of their roles in disease resistance.

Genome-wide identification of drought-responsive microRNAs in two sets of Malus from interspecific hybrid progenies.

Drought stress can negatively impact apple fruit quality and yield. Apple microRNAs (miRNAs) participate in apple tree and fruit development, as well as in biotic stress tolerance; however, it is largely unknown whether these molecules are involved in the drought response. To identify drought-responsive miRNAs in Malus, we first examined the drought stress tolerance of ten F1 progenies of R3 (M. × domestica) × M. sieversii. We performed Illumina sequencing on pooled total RNA from both drought-tolerant and drought-sensitive plants. The sequencing results identified a total of 206 known miRNAs and 253 candidate novel miRNAs from drought-tolerant plants and drought-sensitive plants under control or drought conditions. We identified 67 miRNAs that were differentially expressed in drought-tolerant plants compared with drought-sensitive plants under drought conditions. Under drought stress, 61 and 35 miRNAs were differentially expressed in drought-tolerant and drought-sensitive plants, respectively. We determined the expression levels of seven out of eight miRNAs by stem-loop qPCR analysis. We also predicted the target genes of all differentially expressed miRNAs and identified the expression of some genes. Gene Ontology analyses indicated that the target genes were mainly involved in stimulus response and cellular and metabolic processes. Finally, we confirmed roles of two miRNAs in apple response to mannitol. Our results reveal candidate miRNAs and their associated mRNAs that could be targeted for improving drought tolerance in Malus species, thus providing a foundation for understanding the molecular networks involved in the response of apple trees to drought stress.