First Report of Leaf Rot Caused by Apiospora paraphaeosperma on Lily in Wuhan, China.

Lily (Lilium spp.) is a valuable ornamental bulb flower plant in Liliaceae, and its bulbs have high edible and medicinal value. Compared with bulb propagation of other lilies, seed propagation and short growth period are the most significant characteristics of Lilium×formolongi. In 2023, leaf rot disease (LRD) was observed on approximately 70% of the Lilium×formolongi seedlings sown in an experimental greenhouse in Wuhan, Hubei province, China. Irregular brown water-soaked spots were discovered in the early stages of infected seedlings. Then, spots spread throughout the leaves and caused the leaves to brown, soften, and wilted. A pathogen associated with symptoms was isolated by incubating sterilized leaves on potato dextrose agar plates at 25 ℃ for 2-3 days. Then, a pure single colony was isolated through a single hyphal tip isolation method. The fungal colony was white with abundant aerial mycelium and produced a yellow pigment diffusible into the agar. Microscopically, isolated mycelia were reticulate and pale yellow, while conidia were dark brown, smooth, and spherical, 7.31 to 6.98 × 4.03 to 3.87µm (average 5.44×5.41µm; n=30); oval in lateral view, and had a light stripe in the middle. To identify the species of the fungus at the molecular level, ITS and EF-1α genes were amplified and sequenced using primers ITS1/ITS4 (M Gardes et al. 1993) and 758F/986R (Carbone and Kohn 1999). The BLAST results in GenBank showed that the ITS(OR523578) and EF-1α(PP066842) sequences of LRD shared 99.82% and 99.24% identity with the distinct Apiospora paraphaeosperma strains (GenBank accession MT040110, ON806628.1, respectively). Combined with the morphology of the colony and conidium, the fungus was identified as Ap. paraphaeosperma. In the pathogenicity test, six healthy leaves were inoculated with mycelium disc and then kept in an incubator (22 ℃, 90% humidity, 16h light /8h darkness). The inoculated leaves showed necrosis and wilt symptoms similar to those observed in the greenhouse, while the control leaves were asymptomatic. A re-isolation, morphology identification and DNA sequencing of the fungus confirmed its infection with Ap. paraphaeosperma in Lilium spp. At present, rot caused by Ap. paraphaeosperma has only been reported in Thailand and South Korea, both of which are found on bamboo stems (Hyde et al. 2016; Sun Lul Kwon et al. 2022). As far as we know, this is the first report of leaf rot of lily caused by Ap. paraphaeosperma in China. This report can help identify this disease and further develop effective control measures.

Comparative transcriptome and metabolome analysis revealed diversity in the response of resistant and susceptible rose (Rosa hybrida) varieties to Marssonina rosae.

Rose black spot disease caused by Marssonina rosae is among the most destructive diseases that affects the outdoor cultivation and production of roses; however, the molecular mechanisms underlying the defensive response of roses to M. rosae have not been clarified. To investigate the diversity of response to M. rosae in resistant and susceptible rose varieties, we performed transcriptome and metabolome analyses of resistant (KT) and susceptible (FG) rose varieties and identified differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) in response to M. rosae at different time points. In response to M. rosae, DEGs and DAMs were mainly upregulated compared to the control and transcription factors were concentrated in the WRKY and AP2/ERF families. Gene Ontology analysis showed that the DEGs of FG were mainly enriched in biological processes, such as the abscisic acid-activated signaling pathway, cell wall, and defense response, whereas the DEGs of KT were mainly enriched in Golgi-mediated vesicle transport processes. Kyoto Encyclopedia of Genes and Genomes analysis showed that the DEGs of both varieties were concentrated in plant-pathogen interactions, plant hormone signal transduction, and mitogen-activated protein kinase signaling pathways, with the greatest number of DEGs associated with brassinosteroid (BR) in the plant hormone signal transduction pathway. The reliability of the transcriptome results was verified by qRT-PCR. DAMs of KT were significantly enriched in the butanoate metabolism pathway, whereas DAMs of FG were significantly enriched in BR biosynthesis, glucosinolate biosynthesis, and tryptophan metabolism. Moreover, the DAMs in these pathways were significantly positively correlated with the DEGs. Disease symptoms were aggravated when FG leaves were inoculated with M. rosae after 24-epibrassinolide treatment, indicating that the response of FG to M. rosae involves the BR signaling pathway. Our results provide new insights into the molecular mechanisms underlying rose response to M. rosae and lay a theoretical foundation for formulating rose black spot prevention and control strategies and cultivating resistant varieties.

Gene pyramiding for boosted plant growth and broad abiotic stress tolerance.

Abiotic stresses such as salinity, heat and drought seriously impair plant growth and development, causing a significant loss in crop yield and ornamental value. Biotechnology approaches manipulating specific genes prove to be effective strategies in crop trait modification. The Arabidopsis vacuolar pyrophosphatase gene AVP1, the rice SUMO E3 ligase gene OsSIZ1 and the cyanobacterium flavodoxin gene Fld have previously been implicated in regulating plant stress responses and conferring enhanced tolerance to different abiotic stresses when individually overexpressed in various plant species. We have explored the feasibility of combining multiple favourable traits brought by individual genes to acquire superior plant performance. To this end, we have simultaneously introduced AVP1, OsSIZ1 and Fld in creeping bentgrass. Transgenic (TG) plants overexpressing these three genes performed significantly better than wild type controls and the TGs expressing individual genes under both normal and various abiotic stress conditions, exhibited significantly enhanced plant growth and tolerance to drought, salinity and heat stresses as well as nitrogen and phosphate starvation, which were associated with altered physiological and biochemical characteristics and delicately fine-tuned expression of genes involved in plant stress responses. Our results suggest that AVP1, OsSIZ1 and Fld function synergistically to regulate plant development and plant stress response, leading to superior overall performance under both normal and adverse environments. The information obtained provides new insights into gene stacking as an effective approach for plant genetic engineering. A similar strategy can be extended for the use of other beneficial genes in various crop species for trait modifications, enhancing agricultural production.

The Abundant and Unique Transcripts and Alternative Splicing of the Artificially Autododecaploid London Plane (Platanus × acerifolia).

Transcription and alternative splicing (AS) are now appreciated in plants, but few studies have examined the effects of changing ploidy on transcription and AS. In this study, we showed that artificially autododecaploid plants of London plane (Platanus × acerifolia (Aiton) Willd) had few flowers relative to their hexaploid progenitors. Transcriptome analysis based on full-length Oxford Nanopore Technologies (ONTs) and next-generation sequencing (NGS) revealed that the increased ploidy level in P. × acerifolia led to more transcribed isoforms, accompanied by an increase in the number of isoforms per gene. The functional enrichment of genes indicated that novel genes transcribed specifically in the dodecaploids may have been highly correlated with the ability to maintain genome stability. The dodecaploids showed a higher number of genes with upregulated differentially expressed genes (DEGs) compared with the hexaploid counterpart. The genome duplication of P. × acerifolia resulted mainly in the DEGs involved in basic biological pathways. It was noted that there was a greater abundance of alternative splicing (AS) events and AS genes in the dodecaploids compared with the hexaploids in P. × acerifolia. In addition, a significant difference between the structure and expression of AS events between the hexaploids and dodecaploids of Platanus was found. Of note, some DEGs and differentially spliced genes (DSGs) related to floral transition and flower development were consistent with the few flower traits in the dodecaploids of P. × acerifolia. Collectively, our findings explored the difference in transcription and AS regulation between the hexaploids and dodecaploids of P. × acerifolia and gained new insight into the molecular mechanisms underlying the few-flower phenotype of P. × acerifolia. These results contribute to uncovering the regulatory role of transcription and AS in polyploids and breeding few-flower germplasms.

A transcription factor of the NAC family regulates nitrate-induced legume nodule senescence.

Legumes establish symbioses with rhizobia by forming nitrogen-fixing nodules. Nitrate is a major environmental factor that affects symbiotic functioning. However, the molecular mechanism of nitrate-induced nodule senescence is poorly understood. Comparative transcriptomic analysis reveals an NAC-type transcription factor in Lotus japonicus, LjNAC094, that acts as a positive regulator in nitrate-induced nodule senescence. Stable overexpression and mutant lines of NAC094 were constructed and used for phenotypic characterization. DNA-affinity purification sequencing was performed to identify NAC094 targeting genes and results were confirmed by electrophoretic mobility shift and transactivation assays. Overexpression of NAC094 induces premature nodule senescence. Knocking out NAC094 partially relieves nitrate-induced degradation of leghemoglobins and abolishes nodule expression of senescence-associated genes (SAGs) that contain a conserved binding motif for NAC094. Nitrate-triggered metabolic changes in wild-type nodules are largely affected in nac094 mutant nodules. Induction of NAC094 and its targeting SAGs was almost blocked in the nitrate-insensitive nlp1, nlp4, and nlp1 nlp4 mutants. We conclude that NAC094 functions downstream of NLP1 and NLP4 by regulating nitrate-induced expression of SAGs. Our study fills in a key gap between nitrate and the execution of nodule senescence, and provides a potential strategy to improve nitrogen fixation and stress tolerance of legumes.

Allelic variation of terpene synthases drives terpene diversity in the wild species of the Freesia genus.

Terpene synthases (TPSs) play pivotal roles in conferring the structural diversity of terpenoids, which are mainly emitted from flowers, whereas the genetic basis of the release of floral volatile terpenes remains largely elusive. Though quite similar in sequence, TPS allelic variants still function divergently, and how they drive floral terpene diversity in closely related species remains unknown. Here, TPSs responsible for the floral scent of wild Freesia species were characterized, and the functions of their natural allelic variants, as well as the causal amino acid residues, were investigated in depth. Besides the 8 TPSs previously reported in modern cultivars, 7 additional TPSs were functionally evaluated to contribute to the major volatiles emitted from wild Freesia species. Functional characterization of allelic natural variants demonstrated that allelic TPS2 and TPS10 variants changed the enzymatic capacity while allelic TPS6 variants drove the diversity of floral terpene products. Further residue substitution analysis revealed the minor residues determining the enzyme catalytic activity and product specificity. The clarification of TPSs in wild Freesia species reveals that allelic TPS variants evolved differently to determine the interspecific floral volatile terpenes in the genus and might be used for modern cultivar improvement.

Genome sequencing reveals chromosome fusion and extensive expansion of genes related to secondary metabolism in Artemisia argyi.

Artemisia argyi, as famous as Artemisia annua, is a medicinal plant with huge economic value in the genus of Artemisia and has been widely used in the world for about 3000 years. However, a lack of the reference genome severely hinders the understanding of genetic basis for the active ingredient synthesis of A. argyi. Here, we firstly report a complex chromosome-level genome assembly of A. argyi with a large size of 8.03 Gb, with features of high heterozygosity (2.36%), high repetitive sequences (73.59%) and a huge number of protein-coding genes (279 294 in total). The assembly reveals at least three rounds of whole-genome duplication (WGD) events, including a recent WGD event in the A. argyi genome, and a recent burst of transposable element, which may contribute to its large genome size. The genomic data and karyotype analyses confirmed that A. argyi is an allotetraploid with 34 chromosomes. Intragenome synteny analysis revealed that chromosomes fusion event occurred in the A. argyi genome, which elucidates the changes in basic chromosome numbers in Artemisia genus. Significant expansion of genes related to photosynthesis, DNA replication, stress responses and secondary metabolism were identified in A. argyi, explaining the extensive environmental adaptability and rapid growth characteristics. In addition, we analysed genes involved in the biosynthesis pathways of flavonoids and terpenoids, and found that extensive gene amplification and tandem duplication contributed to the high contents of metabolites in A. argyi. Overall, the reference genome assembly provides scientific support for evolutionary biology, functional genomics and breeding in A. argyi and other Artemisia species.

Characterization of Terpene synthase variation in flowers of wild aquilegia species from Northeastern Asia.

There are several causes for the great diversity in floral terpenes. The terpene products are determined by the catalytic fidelity, efficiency and plasticity of the active sites of terpene synthases (TPSs). However, the molecular mechanism of TPS in catalyzing terpene biosynthesis and its evolutionary fate in wild plant species remain largely unknown. In this study, the functionality of terpene synthases and their natural variants were assessed in two Northeastern Asia endemic columbine species and their natural hybrid. Synoptically, TPS7, TPS8, and TPS9 were highly expressed in these Aquilegia species from the Zuojia population. The in vitro and in vivo enzymatic assays revealed that TPS7 and TPS8 mainly produced (+)-limonene and ß-sesquiphellandrene, respectively, whereas TPS9 produced pinene, similar to the major components released from Aquilegia flowers. Multiple sequence alignment of Aquilegia TPS7 and TPS8 in the Zuojia population revealed amino acid polymorphisms. Domain swapping and amino acid substitution assays demonstrated that 413A, 503I and 529D had impacts on TPS7 catalytic activity, whereas 420G, 538F and 545 L affected the ratio of ß-sesquiphellandrene to ß-bisabolene in TPS8. Moreover, these key polymorphic amino acid residues were found in Aquilegia species from the Changbai Mountain population. Interestingly, amino acid polymorphisms in TPSs were present in individuals with low expression levels, and nonsynonymous mutations could impact the catalytic activity or product specificity of these genes. The results of this study will shed new light on the function and evolution of TPS genes in wild plant species and are beneficial to the modification of plant fragrances.

Homeostatic regulation of flavonoid and lignin biosynthesis in phenylpropanoid pathway of transgenic tobacco.

Flavonoids and lignin consist of a large number of secondarymetabolites which are derived from the phenylpropanoid pathway, and they act as a significant role in plant growth, development, and stress response. However, few reports have documented that how different subbranches of phenylpropanoid metablolic pathway mutually interact. In Arabidopsis, AtCPC (AtCAPRICE) is known to play a negative role in anthocyanin accumulation. Nonetheless, whether AtCPC could control the biosynthesis of lignin is largely unknown. Additionally, whether the RrFLS and RrANR, flavonol synthase and anthocyanidin reductase, from Rosa rugosa regulate different branches of phenylpropanoid pathway is unclear. Here, we performed a series of transgenic experiments with short life cycle tobacco and RNA-Seq analysis. Finally, a series of assays related to biological, physiological, and phenotypic characteristics were undertaken. Our results indicated that ectopic expression of AtCPC in tobacco not only decreased the flavonoid compound accumulation, but also up-regulated several lignin biosynthetic genes, and significantly increased the accumulation of lignin. Our results also revealed that although they respectively improved the flavonol and proanthocyanidin contents, the overexpression of RrFLS and RrANR plays positive roles in lignin biosynthesis in transgenic tobacco plants. Our findings provide a novel insight into the mechanism underlying homeostatic regulation of flavonoid and lignin biosynthesis in phenylpropanoid pathway of plants.

Phytochrome-interacting factors interact with transcription factor CONSTANS to suppress flowering in rose.

As day-neutral (DN) woody perennial plants, the flowering time of roses (Rosa spp.) is assumed to be independent of the photoperiodic conditions; however, light responses of rose plants are not well understood. Chinese rose (Rosa chinensis) plants were grown under two light intensities (low light [LL], 92 µmol·m-2·s-1; or high light [HL], 278 µmol·m-2·s-1), and either with or without an end-of-day far-red (EOD-FR) treatment. Flowering was significantly delayed in the LL condition compared with the HL, but was not affected by EOD-FR treatment. The time until flowering positively corresponded with the mRNA and protein levels of phytochrome-interacting factors (PIFs; RcPIFs). The heterologous expression of RcPIF1, RcPIF3, or RcPIF4 in the Arabidopsis (Arabidopsis thaliana) pifq quadruple mutant partially rescued the mutant's shorter hypocotyl length. Simultaneous silencing of three RcPIFs in R. chinensis accelerated flowering under both LL and HL, with a more robust effect in LL, establishing RcPIFs as flowering suppressors in response to light intensity. The RcPIFs interacted with the transcription factor CONSTANS (RcCO) to form a RcPIFs-RcCO complex, which interfered with the binding of RcCO to the promoter of FLOWERING LOCUS T (RcFT), thereby inhibiting its expression. Furthermore, this inhibition was enhanced when RcPIFs were stabilized by LL, leading to delayed flowering under LL compared with HL. Our results not only revealed another layer of PIF functioning in the flowering of woody perennial plants, but also established a mechanism of light response in DN plants.

A conservative pathway for coordination of cell wall biosynthesis and cell cycle progression in plants.

The mechanism that coordinates cell growth and cell cycle progression remains poorly understood; in particular, whether the cell cycle and cell wall biosynthesis are coordinated remains unclear. Recently, cell wall biosynthesis and cell cycle progression were reported to respond to wounding. Nonetheless, no genes are reported to synchronize the biosynthesis of the cell wall and the cell cycle. Here, we report that wounding induces the expression of genes associated with cell wall biosynthesis and the cell cycle, and that two genes, AtMYB46 in Arabidopsis thaliana and RrMYB18 in Rosa rugosa, are induced by wounding. We found that AtMYB46 and RrMYB18 promote the biosynthesis of the cell wall by upregulating the expression of cell wall-associated genes, and that both of them also upregulate the expression of a battery of genes associated with cell cycle progression. Ultimately, this response leads to the development of curled leaves of reduced size. We also found that the coordination of cell wall biosynthesis and cell cycle progression by AtMYB46 and RrMYB18 is evolutionarily conservative in multiple species. In accordance with wounding promoting cell regeneration by regulating the cell cycle, these findings also provide novel insight into the coordination between cell growth and cell cycle progression and a method for producing miniature plants.

KSN heterozygosity is associated with continuous flowering of Rosa rugosa Purple branch.

Rose (Rosa spp.) plants flower via two contrasting methods: once flowering (OF) and continuous flowering (CF). Purple branch is a rare continuously flowering variety of Rosa rugosa that is extensively cultivated in China. However, the genetic basis of its CF behavior is unknown. We demonstrated that Purple branch is heterozygous for the TFL1 homolog KSN. One KSN allele with a 9 kb Copia insertion was found to be identical to that from continuously flowering Rosa chinensis Old blush. The other allele was found to be a functional wild-type allele. The overall expression of KSN was closely linked to the floral transition, and it was significantly repressed in continuously flowering Purple branch compared with OF Plena. The promoter region of the normal KSN allele was hypermethylated, and histone methylation at H3H4, H3K9, and H3K27 of the KSN gene locus was modified in continuously flowering Purple branch. Silencing of the DNA methyltransferase genes MET1 and CMT3 and the histone methyltransferase gene SUVR5 in Purple branch led to enhanced KSN expression, but silencing of the histone demethylase gene JMJ12 suppressed KSN expression. Therefore, the CF habit of Purple branch may be due to reduced expression of KSN caused by the halved dose and may be associated with epigenetic modifications together with retrotransposon insertions along the chromosome. Our study revealed a novel mechanism underlying the CF behavior of rose plants.

RcMYB84 and RcMYB123 mediate jasmonate-induced defense responses against Botrytis cinerea in rose (Rosa chinensis).

Jasmonates (JAs) are important for pathogen resistance in many plants, but the role of these phytohormones in fungal pathogen resistance in rose is unclear. Here, we determined that exogenous application of methyl jasmonate increased resistance to the important fungal pathogen Botrytis cinerea in Rosa chinensis 'Old blush', whereas silencing the JA biosynthetic pathway gene Allene Oxide Synthase (AOS) and JA co-receptor gene CORONATINE INSENSITIVE 1 (COI1) suppressed this response. Transcriptome profiling identified various MYB transcription factor genes that responded to both JA and B. cinerea treatment. Silencing Ri-RcMYB84/Ri-RcMYB123 increased the susceptibility of rose plants to B. cinerea and inhibited the protective effects of JA treatment, confirming the crucial roles of these genes in JA-induced responses to B. cinerea. JAZ1, a key repressor of JA signaling, directly interacts with RcMYB84 and RcMYB123 to deplete their free pools. The JAZ1-RcMYB84 complex binds to the RcMYB123 promoter via the CAACTG motifs to block its transcription. Upon JA treatment, the expression of RcMYB123 is de-repressed, and free forms of RcMYB84 and RcMYB123 are released due to JAZ1 degradation, thereby activating the defense responses of plants to B. cinerea. These findings shed light on the molecular mechanisms underlying JA-induced pathogen resistance in roses.

Alternate expression of CONSTANS-LIKE 4 in short days and CONSTANS in long days facilitates day-neutral response in Rosa chinensis.

Photoperiodic flowering responses are classified into three major types: long day (LD), short day (SD), and day neutral (DN). The inverse responses to daylength of LD and SD plants have been partly characterized in Arabidopsis and rice; however, the molecular mechanism underlying the DN response is largely unknown. Modern roses are economically important ornamental plants with continuous flowering (CF) features, and are generally regarded as DN plants. Here, RcCO and RcCOL4 were identified as floral activators up-regulated under LD and SD conditions, respectively, in the CF cultivar Rosa chinensis 'Old-Blush'. Diminishing the expression of RcCO or/and RcCOL4 by virus-induced gene silencing (VIGS) delayed flowering time under both SDs and LDs. Interestingly, in contrast to RcCO-silenced plants, the flowering time of RcCOL4-silenced plants was more delayed under SD than under LD conditions, indicating perturbed plant responses to day neutrality. Further analyses revealed that physical interaction between RcCOL4 and RcCO facilitated binding of RcCO to the CORE motif in the promoter of RcFT and induction of RcFT. Taken together, the complementary expression of RcCO in LDs and of RcCOL4 in SDs guaranteed flowering under favorable growth conditions regardless of the photoperiod. This finding established the molecular foundation of CF in roses and further shed light on the underlying mechanisms of DN responses.

CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation.

Legume nodules contain high concentrations of leghemoglobins (Lbs) encoded by several genes. The reason for this multiplicity is unknown. CRISPR/Cas9 technology was used to generate stable mutants of the three Lbs of Lotus japonicus. The phenotypes were characterized at the physiological, biochemical and molecular levels. Nodules of the triple mutants were examined by electron microscopy and subjected to RNA-sequencing (RNA-seq) analysis. Complementation studies revealed that Lbs function synergistically to maintain optimal N2 fixation. The nodules of the triple mutants overproduced superoxide radicals and hydrogen peroxide, which was probably linked to activation of NADPH oxidases and changes in superoxide dismutase isoforms expression. The mutant nodules showed major ultrastructural alterations, including vacuolization, accumulation of poly-ß-hydroxybutyrate and disruption of mitochondria. RNA-seq of c. 20 000 genes revealed significant changes in expression of carbon and nitrogen metabolism genes, transcription factors, and proteinases. Lb-deficient nodules had c. 30-50-fold less heme but similar transcript levels of heme biosynthetic genes, suggesting a post-translational regulatory mechanism of heme synthesis. We conclude that Lbs act additively in nodules and that the lack of Lbs results in early nodule senescence. Our observations also provide insight into the reprogramming of the gene expression network associated with Lb deficiency, probably as a result of uncontrolled intracellular free O2 concentration.

The development of a high-density genetic map significantly improves the quality of reference genome assemblies for rose.

Roses are important woody plants featuring a set of important traits that cannot be investigated in traditional model plants. Here, we used the restriction-site associated DNA sequencing (RAD-seq) technology to develop a high-density linkage map of the backcross progeny (BC1F1) between Rosa chinensis 'Old Blush' (OB) and R. wichuraiana 'Basyes' Thornless' (BT). We obtained 643.63 million pair-end reads and identified 139,834 polymorphic tags that were distributed uniformly in the rose genome. 2,213 reliable markers were assigned to seven linkage groups (LGs). The length of the genetic map was 1,027.425 cM in total with a mean distance of 0.96 cM per marker locus. This new linkage map allowed anchoring an extra of 1.21/23.14 Mb (12.18/44.52%) of the unassembled OB scaffolds to the seven reference pseudo-chromosomes, thus significantly improved the quality of assembly of OB reference genome. We demonstrate that, while this new linkage map shares high collinearity level with strawberry genome, it also features two chromosomal rearrangements, indicating its usefulness as a resource for understanding the evolutionary scenario among Rosaceae genomes. Together with the newly released genome sequences for OB, this linkage map will facilitate the identification of genetic components underpinning key agricultural and biological traits, hence should greatly advance the studies and breeding efforts of rose.

RrMYB5- and RrMYB10-regulated flavonoid biosynthesis plays a pivotal role in feedback loop responding to wounding and oxidation in Rosa rugosa.

Flavonoids play critical roles in plant responses to various stresses. Few studies have been reported on what the mechanism of activating flavonoid biosynthesis in plant responses to wounding and oxidation is. In this study, flavonoid metabolites and many MYB transcript factors from Rosa rugosa were verified to be induced by wounding and oxidation. RrMYB5 and RrMYB10, which belong to PA1- and TT2-type MYB TFs, respectively, showed extremely high induction. Overexpression of RrMYB5 and RrMYB10 resulted in an increased accumulation of proanthocyanidins in R. rugosa and tobacco by promoting the expression of flavonoid structural genes. Transcriptomic analysis of the transgenic plants showed that most genes, involved in wounding and oxidation response and ABA signalling modulation, were up-regulated by the overexpression of RrMYB10, which was very much similar to that observed in RrANR and RrDFR overexpression transgenics. RrMYB5 and RrMYB10 physically interacted and mutually activated each other's expressions. They solely or synergistically activated the different sets of flavonoid pathway genes in a bHLH TF EGL3-independent manner. Eventually, the accumulation of proanthocyanidins enhanced plant tolerance to wounding and oxidative stresses. Therefore, RrMYB5 and RrMYB10 regulated flavonoid synthesis in feedback loop responding to wounding and oxidation in R. rugosa. Our study provides new insights into the regulatory mechanisms of flavonoid biosynthesis by MYB TFs and their essential physiological functions in plant responses to wounding and oxidative stresses.