Indole-3 acetic acid negatively regulates rose black spot disease resistance through antagonizing the salicylic acid signaling pathway via jasmonic acid.

MAIN CONCLUSION: IAA cooperates with JA to inhibit SA and negatively regulates rose black spot disease resistance. Black spot disease caused by the fungus Marssonina rosae is the most prevalent and severe ailment in rose cultivation, leading to the appearance of black spots on leaves and eventual leaf fall, significantly impacting the utilization of roses in gardens. Salicylic acid (SA) and jasmonic acid (JA) are pivotal hormones that collaborate with indole-3 acetic acid (IAA) in regulating plant defense responses; however, the detailed mechanisms underlying the induction of black spot disease resistance by IAA, JA, and SA remain unclear. In this study, transcript analysis was conducted on resistant (R13-54) and susceptible (R12-26) lines following M. rosae infection. In addition, the impact of exogenous interference with IAA on SA- and JA-mediated disease resistance was examined. The continuous accumulation of JA, in synergy with IAA, inhibited activation of the SA signaling pathway in the early infection stage, thereby negatively regulating the induction of effective resistance to black spot disease. IAA administration alleviated the inhibition of SA on JA to negatively regulate the resistance of susceptible strains by further enhancing the synthesis and accumulation of JA. However, IAA did not contribute to the negative regulation of black spot resistance when high levels of JA were inhibited. Virus-induced gene silencing of RcTIFY10A, an inhibitor of the JA signaling pathway, further suggested that IAA upregulation led to a decrease in disease resistance, a phenomenon not observed when the JA signal was inhibited. Collectively, these findings indicate that the IAA-mediated negative regulation of black spot disease resistance relies on activation of the JA signaling pathway.

The Marssonina rosae effector MrSEP43 suppresses rose immunity by targeting the orphan protein RcBROG.

Rose black spot disease, caused by Marssonina rosae (syn. Diplocarpon rosae), is one of the most widespread diseases of field-grown roses worldwide. Pathogens have been found to interfere with or stimulate plant immune response through the secreted effectors. However, the molecular mechanism involved in inhibition of rose immune response by M. rosae effectors remains poorly understood. In this study, we identified the effector MrSEP43, which played a pivotal role in promoting the virulence of M. rosae and enhancing rose susceptibility by reducing callose deposition, H2O2 accumulation, and the expression of defense genes in jasmonic acid signaling pathway. Through Y2H, BiFC, and LUC assays, MrSEP43 was proved to interact with the rose orphan protein RcBROG. RcBROG, which was a positive regulator of defense against M. rosae, enhanced rose resistance by increasing callose deposition, H2O2 accumulation, and expression of RcERF1 in the ethylene signaling pathway. Overall, our findings suggested that the virulence effector MrSEP43 from M. rosae specifically targeted the orphan protein RcBROG to suppress rose immune response to M. rosae. These results provided new insight into how M. rosae manipulated and successfully colonized rose leaves, and were essential for preventing the breakdown of resistance to rose black spot disease.

PfPIN5 promotes style elongation by regulating cell length in Primula forbesii Franch.

BACKGROUND AND AIMS: Style dimorphism is one of the polymorphic characteristics of flowers in heterostylous plants, which have two types of flowers: the pin morph, with long styles and shorter anthers, and the thrum morph, with short styles and longer anthers. The formation of dimorphic styles has received attention in the plant world. Previous studies showed that CYP734A50 in Primula determined style length and limited style elongation and that the brassinosteroid metabolic pathway was involved in regulation of style length. However, it is unknown whether there are other factors affecting the style length of Primula. METHODS: Differentially expressed genes highly expressed in pin morph styles were screened based on Primula forbesii transcriptome data. Virus-induced gene silencing was used to silence these genes, and the style length and anatomical changes were observed 20 days after injection. KEY RESULTS: PfPIN5 was highly expressed in pin morph styles. When PfPIN5 was silenced, the style length was shortened in pin and long-homostyle plants by shortening the length of style cells. Moreover, silencing CYP734A50 in thrum morph plants increased the expression level of PfPIN5 significantly, and the style length increased. The results indicated that PfPIN5, an auxin efflux transporter gene, contributed to regulation of style elongation in P. forbesii. CONCLUSIONS: The results implied that the auxin pathway might also be involved in the formation of styles of P. forbesii, providing a new pathway for elucidating the molecular mechanism of style elongation in P. forbesii.

Integration of chromatin accessibility and gene expression reveals new regulators of cold hardening to enhance freezing tolerance in Prunus mume.

Low temperature is one of the most important abiotic factors limiting the growth, development and geographical distribution of plants. Prunus mume is an attractive woody ornamental plant that blooms in early spring in Beijing. However, the molecular mechanisms underlying cold hardening to enhance freezing tolerance in Prunus genus remains elusive. This study examined the dynamic physiological responses induced by cold hardening, and identified freezing-tolerance genes by RNA-seq and ATAC-seq analyses. Cold hardening elevated the content of soluble substances and enhanced freezing resistance in P. mume. Transcriptome analysis indicated that the candidate differentially expressed genes (DEGs) were those enriched in Ca2+ signalling, mitogen-activated protein kinase (MAPK) cascade, abscisic acid signalling, and inducer of CBF expression 1 (ICE)-C-repeat binding factor (CBF) signalling pathways. The openness of gene chromatin positively correlated with the expression level of these genes. Thirteen motifs were identified in the open chromatin regions in the treatment group subjected to freezing after cold hardening. The chromatin opening of transcription start site at the proximal -177 region of cold-shock protein CS120-like (PmCSL) was markedly increased, while the expression level of PmCSL was significantly up-regulated. Overexpression of PmCSL in Arabidopsis significantly improved the freezing tolerance of transgenic plants. These findings provide new insights into the regulatory mechanism of freezing tolerance to improve breeding of cold-hardy P. mume plants.

Phylogeny of PmCCD Gene Family and Expression Analysis of Flower Coloration and Stress Response in Prunus mume.

The CCD gene family plays a crucial role in the cleavage of carotenoids, converting them into apocarotenoids. This process not only impacts the physiology and development of plants but also enhances their tolerance toward different stresses. However, the character of the PmCCD gene family and its role in ornamental woody Prunus mume remain unclear. Here, ten non-redundant PmCCD genes were identified from the P. mume genome, and their physicochemical characteristics were predicted. According to the phylogenetic tree, PmCCD proteins were classified into six subfamilies: CCD1, CCD4, CCD7, CCD8, NCED and CCD-like. The same subfamily possessed similar gene structural patterns and numbers of conserved motifs. Ten PmCCD genes were concentrated on three chromosomes. PmCCD genes exhibited interspecific collinearity with P. armeniaca and P. persica. Additionally, PmCCD genes had obvious specificity in different tissues and varieties. Compared with white-flowered 'ZLE', PmCCD1 and PmCCD4 genes were low-expressed in 'HJH' with yellow petals, which suggested PmCCD1 and PmCCD4 might be related to the formation of yellow flowers in P. mume. Nine PmCCD genes could respond to NaCl or PEG treatments. These genes might play a crucial role in salt and drought resistance in P. mume. Moreover, PmVAR3 and PmSAT3/5 interacted with PmCCD4 protein in yeast and tobacco leaf cells. This study laid a foundation for exploring the role of the PmCCD gene family in flower coloration and stress response in P. mume.

KNOX Genes Were Involved in Regulating Axillary Bud Formation of Chrysanthemum × morifolium.

Branching is an important agronomic and economic trait in cut chrysanthemums. The axillary meristem (AM) formation of the axillary buds of cut chrysanthemums has a decisive role in its branching characteristics. However, little is known about the regulation mechanism of axillary meristem formation in chrysanthemums at the molecular level. Members of the Homeobox gene family especially genes belonging to the class I KNOX branch play a key role in regulating the axillary bud growth and development processes of plants. In this study, three genes belonging to the class I KNOX branch, CmKNAT1, CmKNAT6, and CmSTM were cloned from chrysanthemums, and their functions in regulating axillary bud formation were examined. The subcellular localization test showed that these three KNOX genes were expressed in the nucleus, so all of them might function as transcription factors. The results of the expression profile analysis showed that these three KNOX genes were highly expressed in the AM formation stage of axillary buds. Overexpression of KNOX genes result in a wrinkled leaf phenotype in tobacco and Arabidopsis, which may be related to the excessive division of leaf cells, resulting in the proliferation of leaf tissue. Furthermore, overexpression of these three KNOX genes enhances the regeneration ability of tobacco leaves, indicating that these three KNOX genes may participate in the regulation of cell meristematic ability, thus promoting the formation of buds. In addition, the results of fluorescence quantitative testing showed that these three KNOX genes may promote the formation of chrysanthemum axillary buds by promoting the cytokinin pathway while inhibiting the auxin and gibberellin pathways. In conclusion, this study demonstrated that CmKNAT1, CmKNAT6, and CmSTM genes were involved in regulating axillary bud formation of Chrysanthemum × morifolium and preliminarily revealed the molecular mechanism of their regulation of AM formation. These findings may provide a theoretical basis and candidate gene resources for genetic engineering breeding of new varieties of cut chrysanthemums without lateral branches.

Genome-Wide Identification of the MAPK and MAPKK Gene Families in Response to Cold Stress in Prunus mume.

Protein kinases of the MAPK cascade family (MAPKKK-MAPKK-MAPK) play an essential role in plant stress response and hormone signal transduction. However, their role in the cold hardiness of Prunus mume (Mei), a class of ornamental woody plant, remains unclear. In this study, we use bioinformatic approaches to assess and analyze two related protein kinase families, namely, MAP kinases (MPKs) and MAPK kinases (MKKs), in wild P. mume and its variety P. mume var. tortuosa. We identify 11 PmMPK and 7 PmMKK genes in the former species and 12 PmvMPK and 7 PmvMKK genes in the latter species, and we investigate whether and how these gene families contribute to cold stress responses. Members of the MPK and MKK gene families located on seven and four chromosomes of both species are free of tandem duplication. Four, three, and one segment duplication events are exhibited in PmMPK, PmvMPK, and PmMKK, respectively, suggesting that segment duplications play an essential role in the expansion and evolution of P. mume and its gene variety. Moreover, synteny analysis suggests that most MPK and MKK genes have similar origins and involved similar evolutionary processes in P. mume and its variety. A cis-acting regulatory element analysis shows that MPK and MKK genes may function in P. mume and its variety's development, modulating processes such as light response, anaerobic induction, and abscisic acid response as well as responses to a variety of stresses, such as low temperature and drought. Most PmMPKs and PmMKKs exhibited tissue-specifific expression patterns, as well as time-specific expression patterns that protect them through cold. In a low-temperature treatment experiment with the cold-tolerant cultivar P. mume 'Songchun' and the cold-sensitive cultivar 'Lve', we find that almost all PmMPK and PmMKK genes, especially PmMPK3/5/6/20 and PmMKK2/3/6, dramatically respond to cold stress as treatment duration increases. This study introduces the possibility that these family members contribute to P. mume's cold stress response. Further investigation is warranted to understand the mechanistic functions of MAPK and MAPKK proteins in P. mume development and response to cold stress.

The chromosome-level genome provides insight into the molecular mechanism underlying the tortuous-branch phenotype of Prunus mume.

Plant with naturally twisted branches is referred to as a tortuous-branch plant, which have extremely high ornamental value due to their zigzag shape and the natural twisting of their branches. Prunus mume is an important woody ornamental plant. However, the molecular mechanism underlying this unique trait in Prunus genus is unknown. Here, we present a chromosome-level genome assembly of the cultivated P. mume var. tortuosa created using Oxford Nanopore combined with Hi-C scaffolding, which resulted in a 237.8 Mb genome assembly being anchored onto eight pseudochromosomes. Molecular dating indicated that P. mume is the most recently differentiated species in Prunus. Genes associated with cell division, development and plant hormones play essential roles in the formation of tortuous branch trait. A putative regulatory pathway for the tortuous branch trait was constructed based on gene expression levels. Furthermore, after transferring candidate PmCYCD genes into Arabidopsis thaliana, we found that seedlings overexpressing these genes exhibited curled rosette leaves. Our results provide insights into the evolutionary history of recently differentiated species in Prunus genus, the molecular basis of stem morphology, and the molecular mechanism underlying the tortuous branch trait and highlight the utility of multi-omics in deciphering the properties of P. mume plant architecture.

HDACs Gene Family Analysis of Eight Rosaceae Genomes Reveals the Genomic Marker of Cold Stress in Prunus mume.

Histone deacetylases (HDACs) play important roles in plant growth, development, and stress response. However, the pattern of how they are expressed in response to cold stress in the ornamental woody plant Prunus mume is poorly understood. Here, we identify 121 RoHDACs from eight Rosaceae plants of which 13 PmHDACs genes are from P. mume. A phylogenetic analysis suggests that the RoHDACs family is classified into three subfamilies, HDA1/RPD3, HD2, and SIR2. We identify 11 segmental duplication gene pairs of RoHDACs and find, via a sequence alignment, that the HDACs gene family, especially the plant-specific HD2 family, has experienced gene expansion and contraction at a recent genome evolution history. Each of the three HDACs subfamilies has its own conserved domains. The expression of PmHDACs in mei is found to be tissue-specific or tissue-wide. RNA-seq data and qRT-PCR experiments in cold treatments suggest that almost all PmHDACs genes-especially PmHDA1/6/14, PmHDT1, and PmSRT1/2-significantly respond to cold stress. Our analysis provides a fundamental insight into the phylogenetic relationship of the HDACs family in Rosaceae plants. Expression profiles of PmHDACs in response to cold stress could provide an important clue to improve the cold hardiness of mei.

Genome-Wide Identification, Evolution, and Expression Analysis of GASA Gene Family in Prunus mume.

The Gibberellic Acid Stimulated Arabidopsis/Gibberellin Stimulated Transcript (GASA/GAST) gene family is a group of plant-specific genes encoding cysteine-rich peptides essential to plant growth, development, and stress responses. Although GASA family genes have been identified in various plant species, their functional roles in Prunus mume are still unknown. In this study, a total of 16 PmGASA genes were identified via a genome-wide scan in Prunus mume and were grouped into three major gene clades based on the phylogenetic tree. All PmGASA proteins possessed the conserved GASA domain, consisting of 12-cysteine residues, but varied slightly in protein physiochemical properties and motif composition. With evolutionary analysis, we observed that duplications and purifying selection are major forces driving PmGASA family gene evolution. By analyzing PmGASA promoters, we detected a number of hormonal-response related cis-elements and constructed a putative transcriptional regulatory network for PmGASAs. To further understand the functional role of PmGASA genes, we analyzed the expression patterns of PmGASAs across different organs and during various biological processes. The expression analysis revealed the functional implication of PmGASA gene members in gibberellic acid-, abscisic acid-, and auxin-signaling, and during the progression of floral bud break in P. mume. To summarize, these findings provide a comprehensive understanding of GASA family genes in P. mume and offer a theoretical basis for future research on the functional characterization of GASA genes in other woody perennials.

The miR156-Targeted SQUAMOSA PROMOTER BINDING PROTEIN (PmSBP) Transcription Factor Regulates the Flowering Time by Binding to the Promoter of SUPPRESSOR OF OVEREXPRESSION OF CO1 (PmSOC1) in Prunus mume.

Prunus mume, a famous perennial ornamental plant and fruit tree in Asia, blooms in winter or early spring in the Yangtze River area. The flowering time directly determines its ornamental and economic value, so it is of great significance to study the molecular mechanism of flowering time. SQUAMOSA PROMOTER BINDING PROTEIN (SBP), often regulated by miR156, is an important flowering regulator, although its function is unknown in P. mume. Here, 11 miR156 precursors were analyzed and located in five chromosomes of the P. mume genome. The expression pattern showed that PmSBP1/6 was negatively correlated with miR156. The promoters of PmSBP1/6 were specifically expressed in the apical meristem. Overexpression of PmSBP1/6 in tobacco promoted flowering and changed the length ratio of pistil and stamen. Moreover, PmSBP1 also affected the number and vitality of pollen and reduced the fertility of transgenic tobacco. Furthermore, ectopic expression of PmSBP1/6 caused up-regulated expression of endogenous SUPPRESSOR OF OVEREXPRESSION OF CO1 (NtSOC1). The yeast-one hybrid assay showed that PmSBP1 was bonded to the promoters of PmSOC1s. In conclusion, a miR156-PmSBP1-PmSOC1s pathway was formed to participate in the regulation of flowering time in P. mume, which provided references for the molecular mechanism of flowering time regulation and molecular breeding of P. mume.

Two Cyc2CL transcripts (Cyc2CL-1 and Cyc2CL-2) may play key roles in the petal and stamen development of ray florets in chrysanthemum.

BACKGROUND: Chrysanthemum morifolium is one of the most popular ornamental crops. The capitulum, which is the main ornamental part of chrysanthemum plants, consists of ligulate marginal ray florets, an attractive corolla (petals), and radially hermaphroditic disc florets, but no stamens. In Asteraceae species, the zygomorphic ray florets evolved from the actinomorphic disc florets. During this process, the zygomorphic ligulate corolla arose and the stamens were aborted. Although molecular genetic research has clarified ray floret development to some extent, the precise molecular mechanism underlying ray floret development in chrysanthemum remained unclear. RESULTS: A CYC2-like gene, Cyc2CL, was cloned from C. morifolium 'Fenditan'. Subsequent analyses revealed that the alternative splicing of Cyc2CL, which occurred in the flower differentiation stage, resulted in the production of Cyc2CL-1 and Cyc2CL-2 in the apical buds. Prior to this stage, only Cyc2CL-1 was produced in the apical buds. A fluorescence in situ hybridization analysis of labeled Cyc2CL-1 and Cyc2CL-2 RNA indicated that Cyc2CL-2 was first expressed in the involucre tissue during the final involucre differentiation stage, but was subsequently expressed in the receptacle and floret primordia as the floral bud differentiation stage progressed. Moreover, Cyc2CL-2 was highly expressed in the inflorescence tissue during the corolla formation stage, and the expression remained high until the end of the floral bud differentiation stage. Furthermore, the overexpression of Cyc2CL-1 and Cyc2CL-2 in transgenic Arabidopsis inhibited stamen and petal development. Therefore, both Cyc2CL-1 and Cyc2CL-2 encode candidate regulators of petal development and stamen abortion and are important for the ray floret development in chrysanthemum. CONCLUSION: In this study, we characterized the alternatively spliced transcripts of the CYC2-like gene that differ subtly regarding expression and function. The data presented herein will be useful for clarifying the regulatory mechanisms associated with the CYC2-like gene and may also be important for identifying the key genes and molecular mechanisms controlling the development of ray florets in chrysanthemum.

CmCYC2-like transcription factors may interact with each other or bind to the promoter to regulate floral symmetry development in Chrysanthemum morifolium.

The complex capitulum of Chrysanthemum morifolium is often comprised of bilaterally symmetrical ray florets and radially symmetrical disc florets. The TCP transcription factor clade CYCLOIDEA2 (CYC2) appears to play a vital role in determining floral symmetry and in regulating floral organ development in Asteraceae. Our previous study identified six CmCYC2 genes from chrysanthemum and showed that CmCYC2c participated in the regulation of ray floret identity. However, the functions of other CmCYC2 genes and the underlying molecular mechanism of CmCYC2-mediated floral development regulation in chrysanthemums have not been elucidated. In this study, we analysed the function of CmCYC2 genes by ectopic expression of CmCYC2 in Arabidopsis. Then, we examined the protein-protein interaction using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Finally, we analysed the protein-DNA interaction using yeast one-hybrid (Y1H) and dual-luciferase reporter assays. We found that ectopic expression of CmCYC2 genes in the Arabidopsis tcp1 mutant changed its floral symmetry and flowering time. Y2H and BiFC assays confirmed three pairs of interactions between CmCYC2 proteins, that is, CmCYC2b-CmCYC2d, CmCYC2b-CmCYC2e and CmCYC2c-CmCYC2d, suggesting that heterodimeric complexes may form between CmCYC2 proteins to increase their functional specificity. The results of Y1H and dual-luciferase reporter assays indicate that CmCYC2c can bind to the promoter of ClCYC2f. Our findings provided clues that CmCYC2-like transcription factors may interact with each other or bind to the promoter to regulate floral symmetry development in C. morifolium. KEY MESSAGE: CmCYC2-like transcription factors may interact with each other or bind to the promoter to regulate floral symmetry development in Chrysanthemum morifolium.

Morpho-physiological integrators, transcriptome and coexpression network analyses signify the novel molecular signatures associated with axillary bud in chrysanthemum.

BACKGROUND: Axillary bud is an important agronomic and economic trait in cut chrysanthemum. Bud outgrowth is an intricate process controlled by complex molecular regulatory networks, physio-chemical integrators and environmental stimuli. Temperature is one of the key regulators of bud's fate. However, little is known about the temperature-mediated control of axillary bud at molecular levels in chrysanthemum. A comprehensive study was designed to study the bud outgrowth at normal and elevated temperature in cut chrysanthemum. Leaf morphology, histology, physiological parameters were studied to correlate the leaf activity with bud morphology, sucrose and hormonal regulation and the molecular controllers. RESULTS: Temperature caused differential bud outgrowth along bud positions. Photosynthetic leaf area, physiological indicators and sucrose utilization were changed considerable due to high temperature. Comparative transcriptome analysis identified a significant proportion of bud position-specific genes.Weighted Gene Co-expression Network Analysis (WGCNA) showed that axillary bud control can be delineated by modules of coexpressed genes; especially, MEtan3, MEgreen2 and MEantiquewhite presented group of genes specific to bud length. A comparative analysis between different bud positions in two temperatures revealed the morpho-physiological traits associated with specific modules. Moreover, the transcriptional regulatory networks were configured to identify key determinants of bud outgrowth. Cell division, organogenesis, accumulation of storage compounds and metabolic changes were prominent during the bud emergence. CONCLUSIONS: RNA-seq data coupled with morpho-physiological integrators from three bud positions at two temperature regimes brings a robust source to understand bud outgrowth status influenced by high temperature in cut chrysanthemum. Our results provide helpful information for elucidating the regulatory mechanism of temperature on axillary bud growth in chrysanthemum.

HpQTL: a geometric morphometric platform to compute the genetic architecture of heterophylly.

Heterophylly, i.e. morphological changes in leaves along the axis of an individual plant, is regarded as a strategy used by plants to cope with environmental change. However, little is known of the extent to which heterophylly is controlled by genes and how each underlying gene exerts its effect on heterophyllous variation. We described a geometric morphometric model that can quantify heterophylly in plants and further constructed an R-based computing platform by integrating this model into a genetic mapping and association setting. The platform, named HpQTL, allows specific quantitative trait loci mediating heterophyllous variation to be mapped throughout the genome. The statistical properties of HpQTL were examined and validated via computer simulation. Its biological relevance was demonstrated by results from a real data analysis of heterophylly in a wood plant, mei (Prunus mume). HpQTL provides a powerful tool to analyze heterophylly and its underlying genetic architecture in a quantitative manner. It also contributes a new approach for genome-wide association studies aimed to dissect the programmed regulation of plant development and evolution.

The genetic architecture of heterochrony as a quantitative trait: lessons from a computational model.

Heterochrony is known as a developmental change in the timing or rate of ontogenetic events across phylogenetic lineages. It is a key concept synthesizing development into ecology and evolution to explore the mechanisms of how developmental processes impact on phenotypic novelties. A number of molecular experiments using contrasting organisms in developmental timing have identified specific genes involved in heterochronic variation. Beyond these classic approaches that can only identify single genes or pathways, quantitative models derived from current next-generation sequencing data serve as a more powerful tool to precisely capture heterochronic variation and systematically map a complete set of genes that contribute to heterochronic processes. In this opinion note, we discuss a computational framework of genetic mapping that can characterize heterochronic quantitative trait loci that determine the pattern and process of development. We propose a unifying model that charts the genetic architecture of heterochrony that perceives and responds to environmental perturbations and evolves over geologic time. The new model may potentially enhance our understanding of the adaptive value of heterochrony and its evolutionary origins, providing a useful context for designing new organisms that can best use future resources.

Screening of optimal reference genes for qRT-PCR and preliminary exploration of cold resistance mechanisms in Prunus mume and Prunus sibirica varieties.

Prunus sibirica and Prunus mume are closely related plant species that differ in cold tolerance. Hybrids of P. sibirica and true mume, belonging to the apricot mei group, inherited strong cold resistance from P. sibirica. These materials are favourable for research on the molecular mechanisms of cold resistance. However, no suitable reference genes have been identified for analysing gene expression patterns between P. sibirica and P. mume. Ten candidate reference genes were assessed, namely, actins (ACT2-1, ACT2-2, ACT2-3, ACT2-4), protein phosphatase 2A-1 (PP2A-1), ubiquitins (UBQ2, UBQ3), ubiquitin extension protein (UBQ1) and tubulins (TUB1, TUB2), with four distinct algorithms (geNorm, NormFinder, BestKeeper and RefFinder). UBQ2 was recognized as the best reference gene in stems and buds across materials (P. sibirica; 'Xiaohong Zhusha', 'Beijing Yudie', and 'Xiao Lve' for true mume; and 'Dan Fenghou', 'Fenghou', and 'Yanxing' for apricot mei) under cold stress. In addition, the temporal and spatial expression patterns of PmCBF6 and PmLEA10 among seven varieties during winter periods were analysed using UBQ2 as a reference gene. The expression differed significantly among cultivars, which may contribute to their differences in cold tolerance. This paper confirmed the strong cold tolerance of apricot mei. And the best internal reference gene suitable for seven varieties was selected: UBQ2. Based on the above results, the expression of PmCBF6 and PmLEA10 genes during wintering in seven varieties was analysed. The molecular mechanisms of cold resistance were found to be possibly different in different varieties of P. sibirica and P. mume.

Selection and validation of appropriate reference genes for gene expression studies in Forsythia.

The qRT-PCR method has been widely used to detect gene expression level in plants, helping to understand the molecular mechanisms. However, there are few researches which focus on the selection of the internal reference genes in Forsythia. To select the appropriate reference genes of Forsythia aimed at qRT-PCR normalization, twelve candidate reference genes were selected from our transcriptome data. Their expression was assessed by RT-PCR analysis in 47 Forsythia samples, including 12 species cultivars, different organs and tissues. GeNorm, NormFinder, and BestKeeper software were used to select the appropriate reference genes, AG and PSY were used to verify the accuracy of the outcome. The results showed that UKN1 was a stable reference gene in leaves of twelve Forsythia germplasms and in different developmental stages of fruits. MTP, ABCT + MTP, and ABCT + MTP + TIP were stable reference genes in different organs. ACT and SDH were stable reference genes in different flower tissues and different developmental stages of the flower buds. When Forsythia plants were stressed with PEG or ABA, SDH + UKN1 + G6PD was the stable reference gene group for qRT-PCR. The results provided the basis for investigating the physiological and biochemical processes of Forsythia related to medicinal and ornamental properties, and drought-resistance in the level of gene expression.

RcAP1, a Homolog of APETALA1, is Associated with Flower Bud Differentiation and Floral Organ Morphogenesis in Rosa chinensis.

Rosa chinensis is one of the most popular flower plants worldwide. The recurrent flowering trait greatly enhances the ornamental value of roses, and is the result of the constant formation of new flower buds. Flower bud differentiation has always been a major topic of interest among researchers. The APETALA1 (AP1) MADS-box (Mcm1, Agamous, Deficiens and SRF) transcription factor-encoding gene is important for the formation of the floral meristem and floral organs. However, research on the rose AP1 gene has been limited. Thus, we isolated AP1 from Rosa chinensis 'Old Blush'. An expression analysis revealed that RcAP1 was not expressed before the floral primordia formation stage in flower buds. The overexpression of RcAP1 in Arabidopsis thaliana resulted in an early-flowering phenotype. Additionally, the virus-induced down-regulation of RcAP1 expression delayed flowering in 'Old Blush'. Moreover, RcAP1 was specifically expressed in the sepals of floral organs, while its expression was down-regulated in abnormal sepals and leaf-like organs. These observations suggest that RcAP1 may contribute to rose bud differentiation as well as floral organ morphogenesis, especially the sepals. These results may help for further characterization of the regulatory mechanisms of the recurrent flowering trait in rose.

Interactions between WUSCHEL- and CYC2-like Transcription Factors in Regulating the Development of Reproductive Organs in Chrysanthemum morifolium.

Chrysanthemum morifolium is a gynomonoecious plant that bears both female zygomorphic ray florets and bisexual actinomorphic disc florets in the inflorescence. This sexual system is quite prevalent in Asteraceae, but poorly understood. CYCLOIDEA (CYC) 2 subclade transcription factors, key regulators of flower symmetry and floret identity in Asteraceae, have also been speculated to function in reproductive organs and could be an entry point for studying gynomonoecy. However, the molecular mechanism is still unclear. On the other hand, the Arabidopsis WUSCHEL (WUS) transcription factor has been proven to play a vital role in the development of reproductive organs. Here, a WUS homologue (CmWUS) in C. morifolium was isolated and characterized. Overexpression of CmWUS in A. thaliana led to shorter siliques and fewer stamens, which was similar to CYC2-like genes reported before. In addition, both CmWUS and CmCYC2 were highly expressed in flower buds during floral organ differentiation and in the reproductive organs at later development stages, indicating their involvement in the development of reproductive organs. Moreover, CmWUS could directly interact with CmCYC2d. Thus, our data suggest a collaboration between CmWUS and CmCYC2 in the regulation of reproductive organ development in chrysanthemum and will contribute to a further understanding of the gynomonoecious sexual system in Asteraceae.