Metabolite and Transcriptome Profiling Analysis Provides New Insights into the Distinctive Effects of Exogenous Melatonin on Flavonoids Biosynthesis in Rosa rugosa.

Although the petals of Rosa rugosa are rich in flavonoids and their bioactivity has a significant impact on human health, the flavonoid content decreases during flower development. In this study, R. rugosa 'Feng hua' was used to investigate the effects of the melatonin foliar spray on enhancing the quality of rose by focusing on major flavonoids. The results showed that the contents of total flavonoids in rose petals at the full bloom stage induced by melatonin obeyed a bell-shaped curve, with a maximum at 0.3 mM, indicating the concentration-dependent up-regulation of flavonoid biosynthesis. In the treatment with 0.3 mM melatonin, metabolomic analyses showed that the concentrations of ten main flavonoids were identified to be increased by melatonin induction, with high levels and increases observed in three flavonols and two anthocyanins. KEGG enrichment of transcriptomic analysis revealed a remarkable enrichment of DEGs in flavonoid and flavonol biosynthesis, such as Rr4CL, RrF3H, and RrANS. Furthermore, functional validation using virus-induced gene silencing technology demonstrated that Rr4CL3 is the crucial gene regulating flavonoid biosynthesis in response to the stimulant of melatonin. This study provides insights into the exogenous melatonin regulation mechanism of biosynthesis of flavonoids, thereby offering potential industrial applications.

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.

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.

The B-box protein BBX19 suppresses seed germination via induction of ABI5.

Seed germination is a fundamental process in the plant life cycle and is regulated by functionally opposing internal and external inputs. Here we explored the role of a negative regulator of photomorphogenesis, a B-box-containing protein (BBX19), as a molecular link between the inhibitory action of the phytohormone abscisic acid (ABA) and the promoting role of light in germination. We show that seeds of BBX19-overexpressing lines, in contrast to those of BBX19 RNA interference lines, display ABA hypersensitivity, albeit independently of elongated hypocotyl 5 (HY5). Moreover, we establish that BBX19 functions neither via perturbation of GA signaling, the ABA antagonistic phytohormone, nor through interference with the DELLA protein germination repressors. Rather, BBX19 functions as an inducer of ABA INSENSITIVE5 (ABI5) by binding to the light-responsive GT1 motifs in the gene promoter. In summary, we identify BBX19 as a regulatory checkpoint, directing diverse developmental processes and tailoring adaptive responses to distinct endogenous and exogenous signals.

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.

Integrated Metabolomics and Transcriptomics Analysis Reveals New Insights into Triterpene Biosynthesis in Rosa rugosa.

Rosa rugosa is highly regarded for its aesthetic and therapeutic qualities. In particular, R. rugosa's flowers are known to produce essential oils containing a mixture of volatile terpenes, phenylpropanoids, and other compounds. Despite this, extensive research exists on volatile terpenes in flowers, while the knowledge of non-volatile terpenes in distinct tissues is still limited. Using UPLC-ESI-MS/MS, a comprehensive analysis of the terpene metabolites in five different tissues of R. rugosa was conducted. These metabolites accumulated in distinct tissues, and the majority of them were triterpenoids. Transcriptome data were collected from five tissues using RNA-seq. Transcriptomics and metabolomics were utilized to evaluate the triterpene biosynthesis pathway, resulting in new insights into its regulation and biosynthesis. The RrOSC10 was identified as a key enzyme in converting 2,3-oxidosqualene into α-amyrin, potentially contributing to the triterpene biosynthesis pathway. Furthermore, the expression of the RrOSC10 gene was upregulated by salinity for 0.5 h and 1 h, with subsequent downregulation at 2 h. This study lays a foundation for future research on the biosynthesis and accumulation of triterpenes in R. rugosa.

The intragenic cis-elements mediate temperature response of RrKSN.

Gene regulation via intragenic sequences is becoming more recognized in many eukaryotes. However, the intragenic sequences mediated gene expressions in response to environmental stimuli have been largely uncharacterized. Here, we showed that the first intron of RrKSN from the Rosa rugosa cultivar 'Purple branch' had a positive effect on RrKSN expression, and the effect depends on its position and orientation. Further analyses revealed that the four adjacent cis-elements (T)CGATT/AATCG(A) within the first intron were critical for the positive regulation, and the RrKSN promotion was significantly suppressed with mutations of these elements. These cis-elements were further evidenced as binding sites for RrARR1, the homologous of Arabidopsis type-B ARABIDOPSIS RESPONSE REGULATOR 1 (ARR1) transcription factor. The first intron-mediated RrKSN expression was enhanced with over-expressing of RrARR1, but abolished with RrARR1 silencing in rose seedlings. Moreover, the expression difference of RrKSN between 16°C and 28°C was eliminated along with RrARR1-silencing. Taken together, these results suggested both RrARR1 and its binding elements are required for the first intron-mediated RrKSN expression in response to varying temperatures. Therefore, our results reveal a unique intragenic regulation mechanism of gene expression by which plants perceive the signal of ambient temperature in rose.

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.

An efficient transient expression system for gene function analysis in rose.

BACKGROUND: Roses are widely used as garden ornamental plants and cut flowers. Rosa chinensis cv 'Old Blush' has been used as a model genotype in rose studies due to its contribution to recurrent flowering and tea scent traits of modern roses. The deficiency of efficient genetic transformation systems is a handicap limiting functional genetics studies of roses. Agrobacterium-mediated transient transformation offers a powerful tool for the characterization of gene function in plants. RESULTS: A convenient and highly efficient Agrobacterium mediated genetic transformation protocol using R. chinensis cv 'Old Blush' seedlings in vitro as an expression system is described in this paper. The most important factor affecting transformation efficiency in this system is seedling age; 3/4-week-old rose shoots with or without roots from sub-culturing are optimal for transformation, requiring no complicated inoculation media, supplements, or carefully tuned plant growth conditions. This transient expression system was successfully applied to analysis of the gene promoter activities, DNA binding capacity of transcription factors, protein-protein interaction in physiological contexts using luciferase as a reporter gene. CONCLUSIONS: This transient transformation system was validated as a robust and efficient platform, thus providing a new option for gene function and signaling pathway investigation in roses and further extending the utility of R. chinensis cv 'Old Blush' as a model plant to study diverse gene function and signaling pathways in Rosaceae.