HortGenome Search Engine, a universal genomic search engine for horticultural crops.

Horticultural crops comprising fruit, vegetable, ornamental, beverage, medicinal and aromatic plants play essential roles in food security and human health, as well as landscaping. With the advances of sequencing technologies, genomes for hundreds of horticultural crops have been deciphered in recent years, providing a basis for understanding gene functions and regulatory networks and for the improvement of horticultural crops. However, these valuable genomic data are scattered in warehouses with various complex searching and displaying strategies, which increases learning and usage costs and makes comparative and functional genomic analyses across different horticultural crops very challenging. To this end, we have developed a lightweight universal search engine, HortGenome Search Engine (HSE; http://hort.moilab.net), which allows for the querying of genes, functional annotations, protein domains, homologs, and other gene-related functional information of more than 500 horticultural crops. In addition, four commonly used tools, including 'BLAST', 'Batch Query', 'Enrichment analysis', and 'Synteny Viewer' have been developed for efficient mining and analysis of these genomic data.

A haplotype-resolved genome assembly of Malus domestica 'Red Fuji'.

The 'Red Fuji' apple (Malus domestica), is one of the most important and popular economic crops worldwide in the fruit industry. Using PacBio HiFi long reads and Hi-C reads, we assembled a high-quality haplotype-resolved genome of 'Red Fuji', with sizes of 668.7 and 668.8 Mb, and N50 sizes of 34.1 and 31.4 Mb. About 97.2% of sequences were anchored in 34 chromosomes. We annotated both haploid genomes, identifying a total of 95,439 protein-coding genes in the two haplotype genomes, with 98% functional annotation. The haplotype-resolved genome of 'Red Fuji' apple stands as a precise benchmark for an array of analyses, such as comparative genomics, transcriptomics, and allelic expression studies. This comprehensive resource is paramount in unraveling variations in allelic expression, advancing quality improvements, and refining breeding efforts.

The chromosome-level genome assembly of the dwarfing apple interstock Malus hybrid 'SH6'.

Malus hybrid 'SH6' (M. honanensis × M. domestica)is a commonly used apple interstock in China, known for its excellent dwarfing characteristics and cold tolerance. In this study, a combined strategy utilizing PacBio HiFi, Hi-C and parental resequencing data were employed to assemble two haploid genomes for 'SH6'. After chromosome anchoring, the final hapH genome size was 596.63 Mb, with a contig N50 of 34.38 Mb. The hapR genome was 649.37 Mb, with a contig N50 of 36.84 Mb. Further analysis predicted that repeated sequences made up 59.69% and 62.52% of the entire genome, respectively. Gene annotations revealed 45,435 genes for hapH and 48,261 genes for hapR. Combined with genomic synteny we suggest that the hapR genome originates from its maternal parent M. domestica cv. Ralls Janet, while the hapH genome comes from its paternal parent, M. honanensis. The assembled genome significantly contributes to the discovery of genes associated with apple dwarfing and the molecular mechanisms governing them.

Hexose/pentose ratio in rhizosphere exudates-mediated soil eutrophic/oligotrophic bacteria regulates the growth pattern of host plant in young apple-aromatic plant intercropping systems.

Introduction: The positive effect of intercropping on host plant growth through plant-soil feedback has been established. However, the mechanisms through which intercropping induces interspecific competition remain unclear. Methods: In this study, we selected young apple trees for intercropping with two companion plants: medium growth-potential Mentha haplocalyx Briq. (TM) and high growth-potential Ageratum conyzoides L. (TA) and conducted mixed intercropping treatment with both types (TMA) and a control treatment of monocropping apples (CT). Results: Our findings revealed that TM increased the under-ground biomass of apple trees and TA and TMA decreased the above-ground biomass of apple trees, with the lowest above-ground biomass of apple trees in TA. The above- and under-ground biomass of intercrops in TA and TMA were higher than those in TM, with the highest in TA, suggesting that the interspecific competition was the most pronounced in TA. TA had a detrimental effect on the photosynthesis ability and antioxidant capacity of apple leaves, resulting in a decrease in above-ground apple biomass. Furthermore, TA led to a reduction in organic acids, alcohols, carbohydrates, and hydrocarbons in the apple rhizosphere soil (FRS) compared to those in both soil bulk (BS) and aromatic plant rhizosphere soil (ARS). Notably, TA caused an increase in pentose content and a decrease in the hexose/pentose (C6/C5) ratio in FRS, while ARS exhibited higher hexose content and a higher C6/C5 ratio. The changes in exudates induced by TA favored an increase in taxon members of Actinobacteria while reducing Proteobacteria in FRS compared to that in ARS. This led to a higher eutrophic/oligotrophic bacteria ratio relative to TM. Discussion: This novel perspective sheds light on how interspecific competition, mediated by root exudates and microbial community feedback, influences plant growth and development.

MdWER interacts with MdERF109 and MdJAZ2 to mediate methyl jasmonate- and light-induced anthocyanin biosynthesis in apple fruit.

Anthocyanin generation in apples (Malus domestica) and the pigmentation that results from it may be caused by irradiation and through administration of methyl jasmonate (MeJA). However, their regulatory interrelationships associated with fruit coloration are not well defined. To determine whether MdERF109, a transcription factor (TF) involved in light-mediated coloration and anthocyanin biosynthesis, has synergistic effects with other proteins, we performed a yeast two-hybrid assessment and identified another TF, MdWER. MdWER was induced by MeJA treatment, and although overexpression of MdWER alone did not promote anthocyanin accumulation co-overexpression with MdERF109 resulted in significantly increase in anthocyanin biosynthesis. MdWER may form a protein complex with MdERF109 to promote anthocyanin accumulation by enhancing combinations between the proteins and their corresponding genes. In addition, MdWER, as a MeJA responsive protein, interacts with the anthocyanin repressor MdJAZ2. Transient co-expression in apple fruit and protein interaction assays allowed us to conclude that MdERF109 and MdJAZ2 interact with MdWER and take part in the production of anthocyanins upon MeJA treatment and irradiation. Our findings validate a role for the MdERF109-MdWER-MdJAZ2 module in anthocyanin biosynthesis and uncover a novel mechanism for how light and MeJA signals are coordinated anthocyanin biosynthesis in apple fruit.

Chromosomal level genome assemblies of two Malus crabapple cultivars Flame and Royalty.

Malus hybrid 'Flame' and Malus hybrid 'Royalty' are representative ornamental crabapples, rich in flavonoids and serving as the preferred materials for studying the coloration mechanism. We generated two sets of high-quality chromosome-level and haplotype-resolved genome of 'Flame' with sizes of 688.2 Mb and 675.7 Mb, and those of 'Royalty' with sizes of 674.1 Mb and 663.6 Mb, all anchored to 17 chromosomes and with a high BUSCO completeness score nearly 99.0%. A total of 47,833 and 47,307 protein-coding genes were annotated in the two haplotype genomes of 'Flame', and the numbers of 'Royalty' were 46,305 and 46,920 individually. The assembled high-quality genomes offer new resources for studying the origin and adaptive evolution of crabapples and the molecular basis of the accumulation of flavonoids and anthocyanins, facilitating molecular breeding of Malus plants.

MPK6-mediated HY5 phosphorylation regulates light-induced anthocyanin accumulation in apple fruit.

Light is known to regulate anthocyanin pigment biosynthesis in plants on several levels, but the significance of protein phosphorylation in light-induced anthocyanin accumulation needs further investigation. In this study, we investigated the dynamics of the apple fruit phosphoproteome in response to light, using high-performance liquid chromatography-tandem mass spectrometry analysis. Among the differentially phosphorylated proteins, the bZIP (basic leucine zipper) transcription factor, HY5, which has been identified as an anthocyanin regulator, was rapidly activated by light treatment of the fruit. We hypothesized that phosphorylated MdHY5 may play a role in light-induced anthocyanin accumulation of apple fruit. Protein interaction and phosphorylation assays showed that mitogen-activated protein kinase MdMPK6 directly interacted with, and activated, MdHY5 via phosphorylation under light conditions, thereby increasing its stability. Consistent with this finding, the suppression of the mitogen-activated protein kinase genes MdMPK6 or MdHY5 resulted in an inhibition of anthocyanin accumulation, and further showed that light-induced anthocyanin accumulation is dependent on MdMPK6 kinase activity, and is required for maximum MdHY5 activity. Under light conditions, active MdMPK6 phosphorylated MdHY5 leading to accumulation of phospho-MdHY5, which enhanced the binding of MdHY5 to its target anthocyanin related genes in fruit. Our findings reveal an MdMPK6-MdHY5 phosphorylation pathway in light-induced anthocyanin accumulation, providing new insights into the regulation of light-induced anthocyanin biosynthesis in apple fruit at both the transcriptional and post-translational levels.

The miR408a-BBP-LAC3/CSD1 module regulates anthocyanin biosynthesis mediated by crosstalk between copper homeostasis and ROS homeostasis during light induction in Malus plants.

INTRODUCTION: The expression of miR408 is affected by copper (Cu) conditions and positively regulates anthocyanin biosynthesis in Arabidopsis. However, the underlying mechanisms by which miR408 regulates anthocyanin biosynthesis mediated by Cu homeostasis and reactive oxygen species (ROS) homeostasis remain unclear in Malus plants. OBJECTIVES: Our study aims to elucidate how miR408a and its target, basic blue protein (BBP) regulate Cu homeostasis and ROS homeostasis, and anthocyanin biosynthesis in Malus plants. METHODS: The roles of miR408a and its target BBP in regulating anthocyanin biosynthesis, Cu homeostasis, and ROS homeostasis were mainly identified in Malus plants. RESULTS: We found that the BBP protein interacted with the copper-binding proteins LAC3 (laccase) and CSD1 (Cu/Zn SOD superoxide dismutase), indicating a potential crosstalk between Cu homeostasis and ROS homeostasis might be mediated by miR408 to regulate the anthocyanin accumulation. Further studies showed that overexpressing miR408a or suppressing BBP transiently significantly increased the expression of genes related to Cu binding and Cu transport, leading to anthocyanin accumulation under light induction in apple fruit and Malus plantlets. Consistently, opposite results were obtained when repressing miR408a or overexpressing BBP. Moreover, light induction significantly increased the expression of miR408a, CSD1, and LAC3, but significantly reduced the BBP expression, resulting in increased Cu content and anthocyanin accumulation. Furthermore, excessive Cu significantly increased the anthocyanin accumulation, accompanied by reduced expression of miR408a and Cu transport genes, and upregulated expression of Cu binding proteins including BBP, LAC3, and CSD1 to maintain the Cu homeostasis and ROS homeostasis in Malus plantlets. CONCLUSION: Our findings provide new insights into the mechanism by which the miR408a-BBP-LAC3/CSD1 module perceives light and Cu signals regulating Cu and ROS homeostasis, ultimately affecting anthocyanin biosynthesis in Malus plants.

Lilac (Syringa oblata) genome provides insights into its evolution and molecular mechanism of petal color change.

Color change during flower opening is common; however, little is understood on the biochemical and molecular basis related. Lilac (Syringa oblata), a well-known woody ornamental plant with obvious petal color changes, is an ideal model. Here, we presented chromosome-scale genome assembly for lilac, resolved the flavonoids metabolism, and identified key genes and potential regulatory networks related to petal color change. The genome assembly is 1.05 Gb anchored onto 23 chromosomes, with a BUSCO score of 96.6%. Whole-genome duplication (WGD) event shared within Oleaceae was revealed. Metabolome quantification identified delphinidin-3-O-rutinoside (Dp3Ru) and cyanidin-3-O-rutinoside (Cy3Ru) as the major pigments; gene co-expression networks indicated WRKY an essential regulation factor at the early flowering stage, ERF more important in the color transition period (from violet to light nearly white), while the MBW complex participated in the entire process. Our results provide a foundation for functional study and molecular breeding in lilac.

Plant Interaction Patterns Shape the Soil Microbial Community and Nutrient Cycling in Different Intercropping Scenarios of Aromatic Plant Species.

Intercropping systems improve the soil nutrient cycle through microbial community activity and then land productivity. However, their interactions mechanism underlying that the mixed aromatic plant species intercropping regulate the soil microbiome and nutrient cycling on the perennial woody orchard is still uncovered. We designed treatments with 0, 1, and 3 aromatic plant species intercropped in two scenarios of clean tillage (T model, T1, T2, and T4) and natural grass (G model, G1, G2, and G4) in apple orchards, and investigated intercrops effects at the branch growing stage (BGS) and fruit development stage (FDS), respectively. Compared with T model, G model in FDS increased alpha diversity of bacterial community and Shannon index fungal community, the relative abundance of dominant taxa, such as Acidobacteria and Actinobacteria, and also the numbers of up and down-regulated OTUs, the most of indices of co-occurrence network in both bacterial and fungal community, and then improved invertase activity and available nitrogen content. Relative to G1, G2 and G4 reduced diversity bacterial community in FDS, the relative abundance of dominant taxa, the most of indices of co-occurrence network, and then improved soil invertase activity and total phosphorus content in soil. Moreover, Shannon index of fungal community, the altered number of OTUs and the most indices of co-occurrence network were higher in G4 than those in G2 in FDS. These changes above in FDS were more markedly than those in BGS, suggesting that chemical diversity of litter from mixed species of aromatic plants in natural grass scenario led to diversity, complexity, and stability of soil microbial community and then nutrient cycling. It provided a novel highlight and method to modulate biocenosis and then improve the soil nutrient cycling.

ROS1 promotes low temperature-induced anthocyanin accumulation in apple by demethylating the promoter of anthocyanin-associated genes.

Low temperature can affect the growth and development of plants through changes in DNA demethylation patterns. Another known effect of low temperature is the accumulation of anthocyanin pigments. However, it is not known whether the two phenomena are linked, specifically, whether DNA demethylation participates in anthocyanin accumulation in response to low-temperature stress. The ROS1 gene is involved in plant DNA demethylation and influences methylation levels in response to low temperature stress. In this study, using RNA sequencing, we detected that the transcription levels of MdROS1 correlate with the anthocyanin content, as well as with those of anthocyanin biosynthesis-related genes in apple (Malus domestica), at low temperatures. Genomic bisulfite sequencing showed that the methylation levels of the promoters of the anthocyanin related genes MdCHS, MdCHI, MdF3'H, MdANS, MdUFGT, and MdMYB10 decreased in apple leaves after low-temperature treatment. Similar expression and methylation results were also found in apple fruit. Transiently silencing MdROS1 in the leaves and fruit of apple cultivars inhibited the accumulation of anthocyanins and led to decreased expression of anthocyanin biosynthetic genes, and the opposite results were detected in MdROS1-overexpressing leaves and fruit. A promoter binding assay showed that the conserved RRD-DME domains of MdROS1 directly bind to the promoters of MdF3'H and MdUFGT. Taken together, these results suggest that ROS1 affects the anthocyanin biosynthetic pathway by decreasing the methylation level of anthocyanin-related gene promoters, thereby increasing their expression and increasing anthocyanin accumulation.

A long noncoding RNA functions in high-light-induced anthocyanin accumulation in apple by activating ethylene synthesis.

Anthocyanin production in apple (Malus domestica) fruit and their consequent coloration can be induced by high-light treatment. The hormone ethylene is also essential for this coloration, but the regulatory relationships that link ethylene and light with anthocyanin-associated coloration are not well defined. In this study, we observed that high-light treatment of apple fruit increased anthocyanin accumulation more than moderate-light treatment did and was the main contributor of induced ethylene production and activation of anthocyanin biosynthesis. A transcriptome study of light-treated apple fruit suggested that a long noncoding RNA (lncRNA), MdLNC610, the corresponding gene of which is physically located downstream from the 1-aminocyclopropane-1-carboxylate oxygenase (ACO) ethylene biosynthesis gene MdACO1, likely affects anthocyanin biosynthesis under high-light treatment. Expression and promoter ß-glucuronidase reporter analyses further showed that MdLNC610 upregulates expression of MdACO1 and so likely participates in high-light-induced ethylene biosynthesis. Overexpression of MdACO1 and MdLNC610 in apple fruit and calli indicated that a major increase in MdLNC610 expression activates MdACO1 expression, thereby causing an increase in ethylene production and anthocyanin levels. These results suggest that MdLNC610 participates in the regulation of high-light-induced anthocyanin production by functioning as a positive regulator to promote MdACO1 expression and ethylene biosynthesis. Our study provides insights into the relationship between mRNA and lncRNA networks in the ethylene biosynthetic pathway and anthocyanin accumulation in apple fruit.

Soil Ventilation Benefited Strawberry Growth via Microbial Communities and Nutrient Cycling Under High-Density Planting.

In order to increase O2 concentration in the rhizosphere and reduce the continuous cropping obstacles under high-density cultivation, ventilation is often used to increase soil aeration. Yet, the effect of ventilation on soil microbial communities and nutrient cycling and, further, the extent to which they influence strawberry growth under greenhouse conditions are still poorly understood. Thus, four treatments-no ventilation + low planting density (LD), ventilation + LD, no ventilation + high planting density (HD), and ventilation + HD-of strawberry "Red cheeks" (Fragaria × ananassa Duch. cv. "Benihopp") were studied in a greenhouse for 3 years. The ventilation pipe (diameter = 10 cm) was buried in the soil at a depth of 15 cm from the surface and fresh air was sent to the root zone through the pipe by a blower. Ten pipes (one pipeline in a row) were attached to a blower. Soil samples were collected using a stainless-steel corer (five-point intra-row sampling) for the nutrient and microbial analyses. The composition and structure of the soil bacterial and fungal communities were analyzed by high-throughput sequencing of the 16S and 18S rRNA genes, and functional profiles were predicted using PICRUSt and FUNGuild, respectively. The results showed that soil ventilation increased the net photosynthetic rate (Pn), transpiration rate (Tr), and water use efficiency (WUE) of strawberry plants across two growth stages [vegetative growth stage (VGS) and fruit development stage (FDS)]. Soil ventilation increased its available nutrient contents, but the available nutrient contents were reduced under the high planting density compared with low planting density. Both the O2 concentration and O2:CO2 ratio were increased by ventilation; these were positively correlated with the relative abundance of Bacilli, Gamma-proteobacteria, Blastocatella, as well as Chytridiomycota and Pezizomycetes. Conversely, ventilation decreased soil CO2 concentration and the abundance of Beta-proteobacteria and Gemmatimonadetes. The greater planting density increased the relative abundance of Acidobacteria (oligotrophic group). Ventilation altered soil temperature and pH along with carbon and nitrogen functional profiles in the VGS (more nitrogen components) and FDS (more carbon components), which benefited strawberry plant growth under high planting density. The practice of soil ventilation provides a strategy to alleviate hypoxia stress and continuous cropping obstacles for improving crop production in greenhouse settings.

The RNA Directed DNA Methylation (RdDM) Pathway Regulates Anthocyanin Biosynthesis in Crabapple (Malus cv. spp.) Leaves by Methylating the McCOP1 Promoter.

The synthesis of anthocyanin pigments in plants is known to be regulated by multiple mechanisms, including epigenetic regulation; however, the contribution of the RNA-directed DNA methylation (RdDM) pathway is not well understood. Here, we used bisulfite sequencing and Real Time (RT)-quantitative (q) PCR to analyze the methylation level of the promoter of constitutively photomorphogenic 1 (McCOP1) from Malus cv. spp, a gene involved in regulating anthocyanin biosynthesis. The CHH methylation level of the McCOP1 promoter was negatively correlated with McCOP1 RNA expression, and inhibiting DNA methylation caused decreased methylation of the McCOP1 promoter and asymmetric cytosine CHH methylation. We observed that the McCOP1 promoter was a direct target of the RdDM pathway argonaute RISC component 4 (McAGO4) protein, which bound to a McCOP1 promoter GGTTCGG site. Bimolecular fluorescence complementation (BIFC) analysis showed that RNA-directed DNA methylation (McRDM1) interacted with McAGO4 and another RdDM protein, domains rearranged methyltransferase 2 (McDRM2), to regulate the CHH methylation of the McCOP1 promoter. Detection of CHH methylation and COP1 gene expression in the Arabidopsis thalianaatago4, atdrm2 and atrdm1 mutants showed that RDM1 is the effector of the RdDM pathway. This was confirmed by silencing McRDM1 in crabapple leaves or apple fruit, which resulted in a decrease in McCOP1 CHH methylation and an increase in McCOP1 transcript levels, as well as in anthocyanin accumulation. In conclusion, these results show that the RdDM pathway is involved in regulating anthocyanin accumulation through CHH methylation of the McCOP1 promoter.

McMYB4 improves temperature adaptation by regulating phenylpropanoid metabolism and hormone signaling in apple.

Temperature changes affect apple development and production. Phenylpropanoid metabolism and hormone signaling play a crucial role in regulating apple growth and development in response to temperature changes. Here, we found that McMYB4 is induced by treatment at 28 °C and 18 °C, and McMYB4 overexpression results in flavonol and lignin accumulation in apple leaves. Yeast one-hybrid (Y1H) assays and electrophoretic mobility shift assays (EMSAs) further revealed that McMYB4 targets the promoters of the flavonol biosynthesis genes CHS and FLS and the lignin biosynthesis genes CAD and F5H. McMYB4 expression resulted in higher levels of flavonol and lignin biosynthesis in apple during growth at 28 °C and 18 °C than during growth at 23 °C. At 28 °C and 18 °C, McMYB4 also binds to the AUX/ARF and BRI/BIN promoters to activate gene expression, resulting in acceleration of the auxin and brassinolide signaling pathways. Taken together, our results demonstrate that McMYB4 promotes flavonol biosynthesis and brassinolide signaling, which decreases ROS contents to improve plant resistance and promotes lignin biosynthesis and auxin signaling to regulate plant growth. This study suggests that McMYB4 participates in the abiotic resistance and growth of apple in response to temperature changes by regulating phenylpropanoid metabolism and hormone signaling.

The long noncoding RNA MdLNC499 bridges MdWRKY1 and MdERF109 function to regulate early-stage light-induced anthocyanin accumulation in apple fruit.

Anthocyanin pigments contribute to plant coloration and are valuable sources of antioxidants in the human diet as components of fruits and vegetables. Their production is known to be induced by light in apple fruit (Malus domestica); however, the underlying molecular mechanism responsible for early-stage light-induced anthocyanin biosynthesis remains unclear. Here, we identified an ethylene response factor (ERF) protein, ERF109, involved in light-induced anthocyanin biosynthesis and found that it promotes coloration by directly binding to anthocyanin-related gene promoters. Promoter::ß-glucuronidase reporter analysis and Hi-C sequencing showed that a long noncoding RNA, MdLNC499, located nearby MdERF109, induces the expression of MdERF109. A W-box cis-element in the MdLNC499 promoter was found to be regulated by a transcription factor, MdWRKY1. Transient expression in apple fruit and stable transformation of apple calli allowed us to reconstruct a MdWRKY1-MdLNC499-MdERF109 transcriptional cascade in which MdWRKY1 is activated by light to increase the transcription of MdLNC499, which in turn induces MdERF109. The MdERF109 protein induces the expression of anthocyanin-related genes and the accumulation of anthocyanins in the early stages of apple coloration. Our results provide a platform for better understanding the various regulatory mechanisms involved in light-induced apple fruit coloration.

The MdMYB16/MdMYB1-miR7125-MdCCR module regulates the homeostasis between anthocyanin and lignin biosynthesis during light induction in apple.

Light induces anthocyanin accumulation and hence decides the coloration of apple fruit. It also plays a key role in regulating the biosynthesis of other secondary metabolites. However, the crosstalk between anthocyanin and lignin metabolism during light induction, which affects the edible quality and visual quality of apple fruit, respectively, have rarely been characterized. In this study, we identified and functionally elucidated the roles of miR7125 and its target, cinnamoyl-coenzyme A reductase gene (CCR), in regulating the homeostasis between anthocyanin and lignin biosynthesis during light induction. Overexpressing miR7125 or inhibiting CCR transiently in apple fruit promoted anthocyanin biosynthesis but reduced lignin production under light-induced conditions. Consistently, opposite results were observed under the background of repressed miR7125 or overexpressed CCR. We found that the repressor MdMYB16 and the activator MdMYB1 bound to the miR7125 promoter. Transient repression of MdMYB16 upregulated miR7125 expression significantly, accompanied by decreased levels of MdCCR transcript, resulting in a reduction in the lignin biosynthesis and an increase in anthocyanin accumulation. However, transient overexpression of MdMYB16 produced the opposite effects to MdMYB16-RNAi. The results reveal a novel mechanism by which the MdMYB16/MdMYB1-miR7125-MdCCR module collaboratively regulates homeostasis between anthocyanin and lignin biosynthesis under light induction in apple.

Apple MPK4 mediates phosphorylation of MYB1 to enhance light-induced anthocyanin accumulation.

Anthocyanins are plant pigments with diverse biological functions that contribute to fruit quality and are beneficial to human health. Anthocyanin accumulation can be influenced by environmental signals, such as light, and plants have developed sophisticated systems to receive and transduce these signals. However, the associated molecular mechanisms are not well understood. In this study, we investigated the potential function of mitogen-activated protein kinases, which are members of the light signaling pathway, during light-induced anthocyanin accumulation in apple (Malus domestica) fruit peels. An antibody array and yeast two-hybrid screen indicated that proteins encoded by two MdMPK4 genes are light-activated and interact with the transcription factor and anthocyanin biosynthesis regulator MdMYB1. A phosphorylation assay showed that the MdMPK4 proteins phosphorylate MdMYB1, thereby increasing its stability under light conditions. Transient MdMPK4 and MdMYB1 overexpression assays further revealed that light-induced anthocyanin accumulation relies on MdMPK4 kinase activity, which is required for maximum MdMYB1 activity. Based on the expression of the chromosome 6 allele MdMPK4-06G under light conditions and the presence of light response elements in the MdMPK4-06G promoter, we concluded that it is more responsive to light than the chromosome 14 allele MdMPK4-14G. These results suggest a potential biotechnological strategy for increasing fruit anthocyanin content via light induction.

Intercropping With Aromatic Plants Increased the Soil Organic Matter Content and Changed the Microbial Community in a Pear Orchard.

Intercropping influences the soil microbiota via litter and root exudate inputs, but the mechanisms by which root exudates mediate the soil microbial community and soil organic matter (SOM) are still unclear. In this study, we selected three aromatic plants (Ocimum basilicum, Tr1; Satureja hortensis, Tr2; Ageratum houstonianum, Tr3) as intercrops that separately grew between rows of pear trees, and no plants were grown as the control in a pear orchard during the spring-summer season for 3 years. The soil from each plot was collected using a stainless-steel corer by five-point sampling between rows of pear trees. The bacterial and fungal communities of the different aromatic intercrops were analyzed by 16S and ITS rRNA gene amplicon sequencing; their functional profiles were predicted by PICRUSt and FUNGuild analyses. The root exudates of the aromatic plants were analyzed by a liquid chromatography-tandem mass spectrometry (LC-MS) system. Compared with the control treatment, all intercropping treatments with aromatic plants significantly increased SOM and soil water content and decreased pH values. The contents of total nitrogen and alkali-hydrolyzable nitrogen in Tr1 and Tr2 were higher than those in Tr3. In Tr3 soil, the relative content of saccharides increased little, whereas the changes in amine (increases) and alcohols (decreases) were rapid. Ageratum houstonianum intercropping decreased the microbial community diversity and significantly influenced the relative abundances of the dominant microbiota (Actinobacteria, Verrucomicrobia, Gemmatimonadetes, Cyanobacteria, Ascomycota, and Basidiomycota) at the phylum, class, and order levels, which increased the assemblage of functional groups (nitrite ammonification, nitrate ammonification, and ureolysis groups). Our study suggested that the main root exudates from aromatic plants shaped the microbial diversity, structure, and functional groups related to the N cycle during SOM mineralization and that intercropping with aromatic plants (especially basil and summer savory) increased N release in the orchard soil.

Transcriptome Analysis Identifies Two Ethylene Response Factors That Regulate Proanthocyanidin Biosynthesis During Malus Crabapple Fruit Development.

Proanthocyanidins (PAs) are a class of flavonoid compounds in plants that play many important roles in pest and disease resistance and are beneficial components of the human diet. The crabapple (Malus) provides an excellent model to study PA biosynthesis and metabolism; therefore, to gain insights into the PA regulatory network in Malus plants, we performed RNA-seq profiling of fruits of the 'Flame' cultivar at five sequential developmental stages. KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis showed that differentially expressed genes (DEGs) related to the functional category 'plant hormone signal transduction' were significantly enriched during fruit development. Further analysis showed that ethylene signal transduction pathway genes or response genes, such as ERS (ethylene response sensor), EIN3 (ETHYLENE INSENSITIVE 3) and ERFs (ethylene response factors), may play an important role in the regulatory network of PA biosynthesis. Additionally, 12 DEGs, including 10 ERFs, 1 MYB, and 1 bHLH transcription factor, associated with PA biosynthesis were identified using WGCNA. The expression patterns of these genes correlated with PA accumulation trends and transcriptome data from qRT-PCR analysis. The expression of RAP2-4 (RELATED TO APETALA 2-4) and RAV1 (related to ABI3/VP1), which belong to the ERF transcription factor family, showed the greatest correlations with PAs accumulation among the 12 identified TFs. Agrobacterium mediated-transient overexpression of the RAP2-4 led to an increase in PA abundance in crabapple leaves and apple fruits, and the opposite results were observed in RAV1-overexpressed crabapple leaves and apple fruits. Moreover, a yeast one-hybrid assay showed that RAP2-4 and RAV1 specifically bound the promoters of the PA biosynthetic genes McLAR1 and McANR2, respectively. These results indicate that RAP2-4 act as an inducer and RAV1 act as a repressor of PA biosynthesis by regulating the expression of the PA biosynthetic genes McLAR1 and McANR2. Taken together, we identified two potential regulators of PA biosynthesis and provide new insights into the ethylene-PA regulatory network.