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Key Message: Analysis of the flower color formation mechanism of 'Rhapsody in Blue' by BF and WF transcriptomes reveals that RhF3'H and RhGT74F2 play a key role in flower color formation. Rosa hybrida has colorful flowers and a high ornamental value. Although rose flowers have a wide range of colors, no blue roses exist in nature, and the reason for this is unclear. In this study, the blue-purple petals (BF) of the rose variety 'Rhapsody in Blue' and the white petals (WF) of its natural mutant were subjected to transcriptome analysis to find genes related to the formation of the blue-purple color. The results showed that the anthocyanin content was significantly higher in BF than in WF. A total of 1077 differentially expressed genes (DEGs) were detected by RNA-Seq analysis, of which 555 were up-regulated and 522 were down-regulated in the WF vs. BF petals. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses of the DEGs revealed that a single gene up-regulated in BF was related to multiple metabolic pathways including metabolic process, cellular process, protein-containing complex, etc. Additionally, the transcript levels of most of the structural genes related to anthocyanin synthesis were significantly higher in BF than in WF. Selected genes were analyzed by qRT-PCR and the results were highly consistent with the RNA-Seq results. The functions of RhF3'H and RhGT74F2 were verified by transient overexpression analyses, and the results confirmed that both affect the accumulation of anthocyanins in 'Rhapsody in Blue'. We have obtained comprehensive transcriptome data for the rose variety 'Rhapsody in Blue'. Our results provide new insights into the mechanisms underlying rose color formation and even blue rose formation., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Key Message: A genome-wide analysis identified 116 NAC genes in rose, including stress-related ones with different expression patterns under drought and salt stress. Silencing of RcNAC091, a member of the ATAF subfamily, decreased dehydration tolerance in rose. The NAC (NAM, ATAF, and CUC) transcription factors (TFs) are plant-specific proteins that regulate various developmental processes and stress responses. However, knowledge of the NAC TFs in rose (Rosa chinensis), one of the most important horticultural crops, is limited. In this study, 116 NAC genes were identified from the rose genome and classified into 16 subfamilies based on protein phylogenetic analysis. Chromosomal mapping revealed that the RcNAC genes were unevenly distributed on the seven chromosomes of rose. Gene structure and motif analysis identified a total of ten conserved motifs, of which motifs 1-7 were highly conserved and present in most rose NACs, while motifs 8-10 were present only in a few subfamilies. Further study of the stress-related RcNACs based on the transcriptome data showed differences in the expression patterns among the organs, at various floral developmental stages, and under drought and salt stress in rose leaves and roots. The stress-related RcNACs possessed cis-regulatory elements (CREs) categorized into three groups corresponding to plant growth and development, phytohormone response, and abiotic and biotic stress response. Reverse transcription-quantitative real-time PCR (RT-qPCR) analysis of 11 representative RcNACs revealed their differential expression in rose leaves and roots under abscisic acid (ABA), polyethylene glycol (PEG), and sodium chloride (NaCl) treatments. Furthermore, the silencing of RcNAC091 verified its role in positively regulating the dehydration stress response. Overall, the present study provides valuable insights into stress-related RcNACs and paves the way for stress tolerance in rose., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Key Message: Genome-wide identification of WD40-like genes reveals a duplication of COP1-like genes, one of the key players involved in regulation of flowering time and photomorphogenesis, with strong functional diversification in Rosaceae. WD40 proteins play crucial roles in a broad spectrum of developmental and physiological processes. Here, we conducted a systematic characterization of this family of genes in Rosa chinensis 'Old Blush' (OB), a founder genotype for modern rose domestication. We identified 187 rose WD40 genes and classified them into 5 clusters and 15 subfamilies with 11 of RcWD40s presumably generated via tandem duplication. We found RcWD40 genes were expressed differentially following stages of vegetative and reproductive development. We detected a duplication of CONSTITUTIVE PHOTOMORPHOGENIC1-like genes in rose (RcCOP1 and RcCOP1L) and other Rosaceae plants. Featuring a distinct expression pattern and a different profile of cis-regulatory-elements in the transcriptional regulatory regions, RcCOP1 seemed being evolutionarily conserved while RcCOP1L did not dimerize with RcHY5 and RcSPA4. Our data thus reveals a functional diversification of COP1-like genes in Rosacaeae plants, and provides a valuable resource to explore the potential function and evolution of WD40-like genes in Rosaceae plants.

Key Message: We genome-wide identified 28 JmjC domain-containing genes, further spatio-temporal expression profiling and genetic analysis defined them as epigenetic regulators in flowering initiation of Rosa chinensis. The JmjC domain-containing histone demethylases play critical roles in maintaining homeostasis of histone methylations, thus are vital for plant growth and development. Genome-wide identification of the JmjC domain-containing genes have been reported in several species, however, no systematic study has been performed in rose plants. In this paper, we identified 28 JmjC domain-containing genes from the newly published genome database of Rosa chinensis. The JmjC domain-containing proteins in R. chinensis were divided into seven groups, KDM3 was the largest group with 13 members, and JmjC domain-only A and KDM5B were the smallest clades both with only one member. Although all the JmjC domain proteins having a conserved JmjC domain, the gene and protein structure experienced differentiation and specification during the evolution, especially in KDM3 clade, one gene (RcJMJ40) was found carrying site deletions for cofactors Fe (II) and α-KG binding which were crucial for demethylase activities, three genes (RcJMJ41, RcJMJ43 and RcJMJ44) had no intron while two of them had tandem JmjC domains. Spatial expression pattern analysis of these JmjC domain-containing genes in different tissues showed most of them were highly expressed in reproductive tissues such as floral meristem and closed flowers than vegetative tissues, demonstrating their important functions in developmental switch from vegetative to reproductive growth of roses. Temporal expression profiling indicated majority of JmjC domain-containing genes from R. chinensis fluctuated along with floral bud differentiation and development, further proving their essential roles in flower organogenesis. VIGS induced silencing of RcJMJ12 led to delayed flowering time, and decreased the expression levels of flowering integrator such as RcFT, RcSOC1, RcFUL, RcLFY and RcAP1, therefore providing the genetic evidence of RcJMJ12 in flowering initiation. Collectively, spatio-temporal expression profiling and genetic analysis defined the JmjC domain-containing genes as important epigenetic regulators in flower development of R. chinensis.

Key Message: H 2 prolonged the vase life and improved the vase quality of cut roses through repressing endogenous ethylene production and alleviating ethylene signal transduction during the entire senescing period. Recently, the application of hydrogen gas (H 2 ) was shown to improve postharvest quality and longevity in perishable horticultural products, but the specific regulation mechanism remains obscure. Here, endogenous ethylene production and the expression of genes in ethylene biosynthesis and signalling pathway were investigated to explore the crosstalk between H 2 and ethylene during the senescence of cut roses. Our results revealed that addition of exogenous ethylene by ethephon accelerated the senescence of cut roses, in which 100 mg L -1 ethephon displayed the most obvious senescent phenotype. While the applied different concentrations (1%, 10%, 50% and 100%) of hydrogen-rich water (HRW) conducted different affects in alleviating the senescence of cut roses, and 1% HRW displayed the best ornamental quality and the longest vase life by reducing ethylene production, supported by the decrease of 1-aminocyclopropene-1-carboxylate (ACC) accumulation, ACC synthase (ACS) and ACC oxidase (ACO) activities, and Rh-ACS3 and Rh-ACO1 expressions in ethylene biosynthesis. In addition, HRW increased the transcripts of ethylene receptor genes Rh-ETR1 at blooming period from day 4 to day 6 and suppressed Rh-ETR3 at senescence phase at day 8 after harvest. Furthermore, the relevant affection of HRW on Rh-ETR1 and Rh-ETR3 expressions still existed when the ethylene production was compromised by adequate addition of exogenous ethylene in HRW-treated cut rose petals, and HRW directly repressed the protein level of Rh-ETR3 in a transient expression assay. Overall, the results suggested that H 2 is involved in neutralizing ethylene-mediated postharvest in cut flowers.

Key Message: Transcriptomic analysis resulted in the upregulation of the genes related to common defense mechanisms for black spot and the downregulation of the genes related to photosynthesis and cell wall modification for powdery mildew. Plant pathogenic fungi successfully colonize their hosts by manipulating the host defense mechanisms, which is accompanied by major transcriptome changes in the host. To characterize compatible plant pathogen interactions at early stages of infection by the obligate biotrophic fungus Podosphaera pannosa, which causes powdery mildew, and the hemibiotrophic fungus Diplocarpon rosae, which causes black spot, we analyzed changes in the leaf transcriptome after the inoculation of detached rose leaves with each pathogen. In addition, we analyzed differences in the transcriptomic changes inflicted by both pathogens as a first step to characterize specific infection strategies. Transcriptomic changes were analyzed using next-generation sequencing based on the massive analysis of cDNA ends approach, which was validated using high-throughput qPCR. We identified a large number of differentially regulated genes. A common set of the differentially regulated genes comprised of pathogenesis-related (PR) genes, such as of PR10 homologs, chitinases and defense-related transcription factors, such as various WRKY genes, indicating a conserved but insufficient PTI [pathogen associated molecular pattern (PAMP) triggered immunity] reaction. Surprisingly, most of the differentially regulated genes were specific to the interactions with either P. pannosa or D. rosae. Specific regulation in response to D. rosae was detected for genes from the phenylpropanoid and flavonoid pathways and for individual PR genes, such as paralogs of PR1 and PR5, and other factors of the salicylic acid signaling pathway. Differently, inoculation with P. pannosa leads in addition to the general pathogen response to a downregulation of genes related to photosynthesis and cell wall modification.

Key Message: Expression analyses revealed that floral transition of Rosa odorata var. gigantea is mainly regulated by VRN1, COLs, DELLA and KSN, with contributions by the effects of phytohormone and starch metabolism. Seasonal plants utilize changing environmental and developmental cues to control the transition from vegetative growth to flowering at the correct time of year. This study investigated global gene expression profiles at different developmental stages of Rosa odorata var. gigantea by RNA-sequencing, combined with phenotypic characterization and physiological changes. Gene ontology enrichment analysis of the differentially expressed genes (DEGs) between four different developmental stages (vegetative meristem, pre-floral meristem, floral meristem and secondary axillary buds) indicated that DNA methylation and the light reaction played a large role in inducing the rose floral transition. The expression of SUF and FLC, which are known to play a role in delaying flowering until vernalization, was down-regulated from the vegetative to the pre-floral meristem stage. In contrast, the expression of VRN1, which promotes flowering by repressing FLC expression, increased. The expression of DELLA proteins, which function as central nodes in hormone signaling pathways, and probably involve interactions between GA, auxin, and ABA to promote the floral transition, was well correlated with the expression of floral integrators, such as AGL24, COL4. We also identified DEGs associated with starch metabolism correlated with SOC1, AGL15, SPL3, AGL24, respectively. Taken together, our results suggest that vernalization and photoperiod are prominent cues to induce the rose floral transition, and that DELLA proteins also act as key regulators. The results summarized in the study on the floral transition of the seasonal rose lay a foundation for further functional demonstration, and have profound economic and ornamental values.

Homeobox (HB) proteins are important transcription factors that regulate the developmental decisions of eukaryotes. WUSCHEL-related homeobox (WOX) transcription factors, known as a plant-specific HB family, play a key role in plant developmental processes. Our previous work has indicated that rhizoids are induced by auxin in rose (Rosa spp.), which acts as critical part of an efficient plant regeneration system. However, the function of WOX genes in auxin-induced rhizoid formation remains unclear. Here, we isolated and characterized a WUSCHEL-related homeobox gene from Rosa canina, RcWOX1, containing a typical homeodomain with 65 amino acid residues. Real-time reverse transcription PCR (qRT-PCR) analysis revealed that RcWOX1 was expressed in the whole process of callus formation and in the early stage of rhizoid formation. Moreover, its expression was induced by auxin treatment. In Arabidopsis transgenic lines expressing the RcWOX1pro::GUS and 35S::GFP-RcWOX1, RcWOX1 was specifically expressed in roots and localized to the nucleus. Overexpression of RcWOX1 in Arabidopsis increased lateral root density and induced upregulation of PIN1 and PIN7 genes. Therefore, we postulated that RcWOX1 is a functional transcription factor that plays an essential role in auxin-induced rhizoid formation.

The late embryogenesis abundant (LEA) protein family is a large protein family that is closely associated with resistance to abiotic stresses in many organisms, such as plants, bacteria and animals. In this study, we isolated a LEA gene, RcLEA, which was cytoplasm-localized, from Rosa chinensis. RcLEA was found to be induced by high temperature through RT-PCR. Overexpression of RcLEA in Escherichia coli improved its growth performance compared with the control under high temperature, low temperature, NaCl and oxidative stress conditions. RcLEA was also overexpressed in Arabidopsis thaliana. The transgenic Arabidopsis showed better growth after high and low temperature treatment and exhibited less peroxide according to 3, 3-diaminobenzidine staining. However, RcLEA did not improve the tolerance to NaCl or osmotic stress in Arabidopsis. In vitro analysis showed that RcLEA was able to prevent the freeze-thaw-induced inactivation or heat-induced aggregation of various substrates, such as lactate dehydrogenase and citrate synthase. It also protected the proteome of E. coli from denaturation when the proteins were heat-shocked or subjected to acidic conditions. Furthermore, bimolecular fluorescence complementation assays suggested that RcLEA proteins function in a complex manner by making the form of homodimers.

Aquaporins (AQPs) are multifunctional membrane channels and facilitate the transport of water across plant cell membranes. Among the plant AQPs, plasma membrane intrinsic proteins (PIPs), which cluster in two phylogenetic groups (PIP1 and PIP2), play a key role in plant growth. Our previous work has indicated that RhPIP2;1, a member of PIP2, is involved in ethylene-regulated cell expansion of rose petals. However, whether PIP1s also play a role in petal expansion is still unclear. Here, we identified RhPIP1;1, a PIP1 subfamily member, from 18 PIPs assemble transcripts in rose microarray database responsive to ethylene. RhPIP1;1 was rapidly and significantly down-regulated by ethylene treatment. RhETRs-silencing also clearly decreased the expression of RhPIP1;1 in rose petals. The activity of the RhPIP1;1 promoter was repressed by ethylene in rosettes and roots of Arabidopsis. RhPIP1;1 is mainly localized on endoplasmic reticulum and plasma membrane. We demonstrated that RhPIP1;1-silencing significantly inhibited the expansion of petals with decreased petal size and cell area, as well as reduced fresh weight when compared to controls. Expression of RhPIP1;1 in Xenopus oocytes indicated that RhPIP1;1 was inactive in terms of water transport, while coexpression of RhPIP1;1 with the functional RhPIP2;1 led to a significant increase in plasma membrane permeability. Yeast growth, β-Galactosidase activity, bimolecular fluorescence complementation, and colocalization assay proved existence of the interaction between RhPIP1;1 and RhPIP2;1. We argue that RhPIP1;1 plays an important role in ethylene-regulated petal cell expansion, at least partially through the interaction with RhPIP2;1.

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein known to contain the unusual amino acid hypusine. It is a highly conserved protein found in all eukaryotic organisms. Although originally identified as a translation initiation factor, recent studies suggest that eIF5A is mainly involved in translation elongation, mRNA turnover and decay, cell proliferation, and programmed cell death. However, the precise cellular function of eIF5A remains largely unknown, especially in plants. Here, we report the identification and characterization of RceIF5A from Rosa chinensis. RceIF5A expression is up-regulated in Rosa chinensis under high temperature, and oxidative and osmotic stress conditions. We produced transgenic Arabidopsis that constitutively enhanced or suppressed expression of RceIF5A. The RceIF5A over-expression plants exhibited increased resistance to heat, and oxidative and osmotic stresses, while the suppressed expression plants (three AteIF5A isoforms in Arabidopsis were down-regulated) showed more susceptibility to these stresses. These results reveal a new physiological role for eIF5A in plants and contribute to the elucidation of the molecular mechanisms involved in the stress response pathway.

Rose flowers, like flowers and fruits of many other plants, produce and emit the aromatic volatiles 2-phenylacetaldehyde (PAA) and 2-phenylethylalchohol (PEA) which have a distinctive flowery/rose-like scent. Previous studies in rose have shown that, similar to petunia flowers, PAA is formed from L: -phenylalanine via pyridoxal-5'-phosphate-dependent L: -aromatic amino acid decarboxylase. Here we demonstrate the use of a Saccharomyces cerevisiae aro10 mutant to functionally characterize a Rosa hybrida cv. Fragrance Cloud sequence (RhPAAS) homologous to petunia phenylacetaldehyde synthase (PhPAAS). Volatile headspace analysis of the aro10 knockout strain showed that it produces up to eight times less PAA and PEA than the WT. Expression of RhPAAS in aro10 complemented the yeast's mutant phenotype and elevated PAA levels, similar to petunia PhPAAS. PEA production levels were also enhanced in both aro10 and WT strains transformed with RhPAAS, implying an application for metabolic engineering of PEA biosynthesis in yeast. Characterization of spatial and temporal RhPAAS transcript accumulation in rose revealed it to be specific to floral tissues, peaking in mature flowers, i.e., coinciding with floral scent production and essentially identical to other rose scent-related genes. RhPAAS transcript, as well as PAA and PEA production in flowers, displayed a daily rhythmic behavior, reaching peak levels during the late afternoon hours. Examination of oscillation of RhPAAS transcript levels under free-running conditions suggested involvement of the endogenous clock in the regulation of RhPAAS expression in rose flowers.

Esters are important contributors to the aroma of numerous flowers and fruits. Acetate esters such as geranyl acetate, phenylethyl acetate and benzyl acetate are generated as a result of the action of alcohol acetyltransferases (AATs). Numerous homologous AATs from various plants have been characterized using in-vitro assays. To study the function of rose alcohol acetyltransferase (RhAAT) in planta, we generated transgenic petunia plants expressing the rose gene under the control of a CaMV-35S promoter. Although the preferred substrate of RhAAT in vitro is geraniol, in transgenic petunia flowers, it used phenylethyl alcohol and benzyl alcohol to produce the corresponding acetate esters, not generated by control flowers. The level of benzyl alcohol emitted by the flowers of different transgenic lines was ca. three times higher than that of phenylethyl alcohol, which corresponded to the ratio between the respective products, i.e. ca. three times more benzyl acetate than phenylethyl acetate. Feeding of transgenic petunia tissues with geraniol or octanol led to the production of their respective acetates, suggesting the dependence of volatile production on substrate availability.