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.

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.

A 49-bp deletion of PmAP2L results in a double flower phenotype in Prunus mume.

The double flower is an important trait with substantial ornamental value. While mutations in PETALOSA TOE-type or AG (AGAMOUS) genes play a crucial role in enhancing petal number in ornamental plants, the complete mechanism underlying the formation of double flowers remains to be fully elucidated. Through the application of bulked segregant analysis (BSA), we identified a novel gene, APETALA2-like (PmAP2L), characterized by a 49-bp deletion in double-flowered Prunus mume. ß-Glucuronidase (GUS) staining and luciferase reporter assays confirmed that the 49-bp deletion in PmAP2L reduced its binding with Pmu-miRNA172a. Phylogenetic analysis and microsynteny analysis suggested that PmAP2L was not a PETALOSA TOE-type gene, and it might be a new gene controlling the formation of double flower in P. mume. Subsequently, overexpression of PmAP2L-D in tobacco led to a significant rise in the number of stamens and the conversion of stamens to petals. Furthermore, silencing of the homologue of RC5G0530900 in rose significantly reduced the number of petals. Using transient gene expression in P. mume flower buds, we determined the functional differences between PmAP2L-D and PmAP2-S in controlling flower development. Meanwhile, DNA-affinity purification sequencing (DAP-seq), yeast hybrid assays and luciferase reporter assays indicated that PmAP2L negatively regulated the floral organ identity genes by forming a repressor complex with PmTPL and PmHDA6/19. Overall, these findings indicate that the variation in PmAP2L is associated with differences in the regulation of genes responsible for floral organ identity, providing new insights into the double-flower trait and double-flower breeding in plants.

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.

Genome-Wide Identification of Callose Synthase Family Genes and Their Expression Analysis in Floral Bud Development and Hormonal Responses in Prunus mume.

Callose is an important polysaccharide composed of beta-1,3-glucans and is widely implicated in plant development and defense responses. Callose synthesis is mainly catalyzed by a family of callose synthases, also known as glucan synthase-like (GSL) enzymes. Despite the fact that GSL family genes were studied in a few plant species, their functional roles have not been fully understood in woody perennials. In this study, we identified total of 84 GSL genes in seven plant species and classified them into six phylogenetic clades. An evolutionary analysis revealed different modes of duplication driving the expansion of GSL family genes in monocot and dicot species, with strong purifying selection constraining the protein evolution. We further examined the gene structure, protein sequences, and physiochemical properties of 11 GSL enzymes in Prunus mume and observed strong sequence conservation within the functional domain of PmGSL proteins. However, the exon-intron distribution and protein motif composition are less conservative among PmGSL genes. With a promoter analysis, we detected abundant hormonal responsive cis-acting elements and we inferred the putative transcription factors regulating PmGSLs. To further understand the function of GSL family genes, we analyzed their expression patterns across different tissues, and during the process of floral bud development, pathogen infection, and hormonal responses in Prunus species and identified multiple GSL gene members possibly implicated in the callose deposition associated with bud dormancy cycling, pathogen infection, and hormone signaling. In summary, our study provides a comprehensive understanding of GSL family genes in Prunus species and has laid the foundation for future functional research of callose synthase genes in perennial trees.

Genome assembly and resequencing analyses provide new insights into the evolution, domestication and ornamental traits of crape myrtle.

Crape myrtle (Lagerstroemia indica) is a globally used ornamental woody plant and is the representative species of Lagerstroemia. However, studies on the evolution and genomic breeding of L. indica have been hindered by the lack of a reference genome. Here we assembled the first high-quality genome of L. indica using PacBio combined with Hi-C scaffolding to anchor the 329.14-Mb genome assembly into 24 pseudochromosomes. We detected a previously undescribed independent whole-genome triplication event occurring 35.5 million years ago in L. indica following its divergence from Punica granatum. After resequencing 73 accessions of Lagerstroemia, the main parents of modern crape myrtle cultivars were found to be L. indica and L. fauriei. During the process of domestication, genetic diversity tended to decrease in many plants, but this was not observed in L. indica. We constructed a high-density genetic linkage map with an average map distance of 0.33 cM. Furthermore, we integrated the results of quantitative trait locus (QTL) using genetic mapping and bulk segregant analysis (BSA), revealing that the major-effect interval controlling internode length (IL) is located on chr1, which contains CDL15, CRG98, and GID1b1 associated with the phytohormone pathways. Analysis of gene expression of the red, purple, and white flower-colour flavonoid pathways revealed that differential expression of multiple genes determined the flower colour of L. indica, with white flowers having the lowest gene expression. In addition, BSA of purple- and green-leaved individuals of populations of L. indica was performed, and the leaf colour loci were mapped to chr12 and chr17. Within these intervals, we identified MYB35, NCED, and KAS1. Our genome assembly provided a foundation for investigating the evolution, population structure, and differentiation of Myrtaceae species and accelerating the molecular breeding of L. indica.

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.

Integrative Analysis of Metabolome and Transcriptome Revealed Lutein Metabolism Contributed to Yellow Flower Formation in Prunus mume.

Prunus mume is a famous ornamental woody tree with colorful flowers. P. mume with yellow flowers is one of the most precious varieties. Regretfully, metabolites and regulatory mechanisms of yellow flowers in P. mume are still unclear. This hinders innovation of flower color breeding in P. mume. To elucidate the metabolic components and molecular mechanisms of yellow flowers, we analyzed transcriptome and metabolome between 'HJH' with yellow flowers and 'ZLE' with white flowers. Comparing the metabolome of the two varieties, we determined that carotenoids made contributions to the yellow flowers rather than flavonoids. Lutein was the key differential metabolite to cause yellow coloration of 'HJH'. Transcriptome analysis revealed significant differences in the expression of carotenoid cleavage dioxygenase (CCD) between the two varieties. Specifically, the expression level of PmCCD4 was higher in 'ZLE' than that in 'HJH'. Moreover, we identified six major transcription factors that probably regulated PmCCD4 to affect lutein accumulation. We speculated that carotenoid cleavage genes might be closely related to the yellow flower phenotype in P. mume. Further, the coding sequence of PmCCD4 has been cloned from the 'HJH' petals, and bioinformatics analysis revealed that PmCCD4 possessed conserved histidine residues, ensuring its enzymatic activity. PmCCD4 was closely related to PpCCD4, with a homology of 98.16%. Instantaneous transformation analysis in petal protoplasts of P. mume revealed PmCCD4 localization in the plastid. The overexpression of PmCCD4 significantly reduced the carotenoid content in tobacco plants, especially the lutein content, indicating that lutein might be the primary substrate for PmCCD4. We speculated that PmCCD4 might be involved in the cleavage of lutein in plastids, thereby affecting the formation of yellow flowers in P. mume. This work could establish a material and molecular basis of molecular breeding in P. mume for improving the flower color.

Whole-Genome Identification of Regulatory Function of CDPK Gene Families in Cold Stress Response for Prunus mume and Prunus mume var. Tortuosa.

Calcium-dependent protein kinases (CDPK) are known to mediate plant growth and development and respond to various environmental changes. Here, we performed whole-genome identification of CDPK families in cultivated and wild mei (Prunus mume). We identified 14 and 17 CDPK genes in P. mume and P. mume var. Tortuosa genomes, respectively. All 270 CPDK proteins were classified into four clade, displaying frequent homologies between these two genomes and those of other Rosaceae species. Exon/intron structure, motif and synteny blocks were conserved between P. mume and P. mume var. Tortuosa. The interaction network revealed all PmCDPK and PmvCDPK proteins is interacted with respiratory burst oxidase homologs (RBOHs) and mitogen-activated protein kinase (MAPK). RNA-seq data analysis of cold experiments show that cis-acting elements in the PmCDPK genes, especially PmCDPK14, are associated with cold hardiness. Our results provide and broad insights into CDPK gene families in mei and their role in modulating cold stress response in plants.

Corrigendum: Genome-wide identification of Aux/IAA gene family and their expression analysis in Prunus mume.

[This corrects the article DOI: 10.3389/fgene.2022.1013822.].

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.

FsHemF is involved in the formation of yellow Forsythia leaves by regulating chlorophyll synthesis in response to light intensity.

The leaves of Forsythia koreana 'Suwon Gold' are yellow under natural light condition and can revert to green when the light intensity is reduced. To understand the molecular mechanism of leaf color changes in response to light intensity, we compared the chlorophyll content and precursor content between yellow- and green-leaf Forsythia under shade and light-recovery conditions. We identified the conversion of coproporphyrin III (Coprogen III) to protoporphyrin IX (Proto IX) as the primary rate-limiting step of chlorophyll biosynthesis in yellow-leaf Forsythia. Further analysis of the activity of the enzymes that catalyze this step and the expression pattern of the chlorophyll biosynthesis-related genes under different light intensities revealed that the negatively regulated expression of FsHemF by light intensity was the major cause affecting the leaf color change in response to light intensity in yellow-leaf Forsythia. To further understand the cause of differential expression pattern of FsHemF in yellow- and green-leaf lines, we compared the coding sequence and promoter sequence of FsHemF between yellow- and green-leaf Forsythia. We found that one G-box light-responsive cis-element was absent in the promoter region of green-leaf lines. To investigate the functional role of FsHemF, we performed virus-induced gene silencing (VIGS) of FsHemF in green-leaf Forsythia, which leads to yellowing leaf veins, decreased chlorophyll b content, and inhibition of chlorophyll biosynthesis. The results will assist in elucidating the mechanism of yellow-leaf Forsythia in response to light intensity.

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.

Multiomics analysis reveals the mechanisms underlying the different floral colors and fragrances of Rosa hybrida cultivars.

The color and fragrance of rose flowers affect their commercial value. However, several rose varieties with new floral colors developed by the bud mutation method lost their fragrance during the breeding process, raising the question: Is there a relationship between floral color and aroma traits? Rose cultivar 'Yellow Island' (YI) with intensely aroma and yellow petals, while its bud mutant 'Past Feeling' (PF) with light aroma and pink petals mixing some yellow, two cultivars were used to explore this question using multiomics approaches. We investigated the genomic polymorphisms between PF and YI by whole-genome resequencing. 71 differentially abundant metabolites and 155 related differentially expressed genes identified in petals between PF and YI. From this, we constructed a model of metabolic changes affecting floral color and fragrance integrating shikimate, terpenoid, carotenoid, and green leaf volatile metabolites and predicted the associated key genes and transcription factors. This study provides a reference for understanding the molecular mechanism of variation in rose floral color and aroma traits.

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.

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.

Genome-wide identification of Aux/IAA gene family and their expression analysis in Prunus mume.

AUXIN/INDOLE ACETIC ACIDs (Aux/IAAs), an early auxin-responsive gene family, is important for plant growth and development. To fully comprehend the character of Aux/IAA genes in woody plants, we identified 19 PmIAA genes in Prunus mume and dissected their protein domains, phylogenetic relationship, gene structure, promoter, and expression patterns during floral bud flushing, auxin response, and abiotic stress response. The study showed that PmIAA proteins shared conserved Aux/IAA domain, but differed in protein motif composition. 19 PmIAA genes were divided into six groups (Groups Ⅰ to Ⅵ) based on phylogenetic analysis. The gene duplication analysis showed that segmental and dispersed duplication greatly influenced the expansion of PmIAA genes. Moreover, we identified and classified the cis-elements of PmIAA gene promoters and detected elements that are related to phytohormone responses and abiotic stress responses. With expression pattern analysis, we observed the auxin-responsive expression of PmIAA5, PmIAA17, and PmIAA18 in flower bud, stem, and leaf tissues. PmIAA5, PmIAA13, PmIAA14, and PmIAA18 were possibly involved in abiotic stress responses in P. mume. In general, these results laid the theoretical foundation for elaborating the functions of Aux/IAA genes in perennial woody plant development.

Comparative transcriptome analysis identified ChlH and POLGAMMA2 in regulating yellow-leaf coloration in Forsythia.

Leaf color is one of the most important features for plants used for landscape and ornamental purposes. However, the regulatory mechanism of yellow leaf coloration still remains elusive in many plant species. To understand the complex genetic mechanism of yellow-leaf Forsythia, we first compared the pigment content and leaf anatomical structure of yellow-leaf and green-leaf accessions derived from a hybrid population. The physiological and cytological analyses demonstrated that yellow-leaf progenies were chlorophyll deficient with defected chloroplast structure. With comparative transcriptome analysis, we identified a number of candidate genes differentially expressed between yellow-leaf and green-leaf Forsythia plants. Among these genes, we further screened out two candidates, ChlH (magnesium chelatase Subunit H) and POLGAMMA2 (POLYMERASE GAMMA 2), with consistent relative-expression pattern between different colored plants. To verify the gene function, we performed virus-induced gene silencing assays and observed yellow-leaf phenotype with total chlorophyll content reduced by approximately 66 and 83% in ChlH-silenced and POLGAMMA2-silenced plants, respectively. We also observed defected chloroplast structure in both ChlH-silenced and POLGAMMA2-silenced Forsythia. Transient over-expression of ChlH and POLGAMMA2 led to increased chlorophyll content and restored thylakoid architecture in yellow-leaf Forsythia. With transcriptome sequencing, we detected a number of genes related to chlorophyll biosynthesis and chloroplast development that were responsive to the silencing of ChlH and POLGAMMA2. To summarize, ChlH and POLGAMMA2 are two key genes that possibly related to yellow-leaf coloration in Forsythia through modulating chlorophyll synthesis and chloroplast ultrastructure. Our study provided insights into the molecular aspects of yellow-leaf Forsythia and expanded the knowledge of foliage color regulation in woody ornamental plants.

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.

Integrated transcriptome and small RNA sequencing in revealing miRNA-mediated regulatory network of floral bud break in Prunus mume.

MicroRNAs is one class of small non-coding RNAs that play important roles in plant growth and development. Though miRNAs and their target genes have been widely studied in many plant species, their functional roles in floral bud break and dormancy release in woody perennials is still unclear. In this study, we applied transcriptome and small RNA sequencing together to systematically explore the transcriptional and post-transcriptional regulation of floral bud break in P. mume. Through expression profiling, we identified a few candidate genes and miRNAs during different developmental stage transitions. In total, we characterized 1,553 DEGs associated with endodormancy release and 2,084 DEGs associated with bud flush. Additionally, we identified 48 known miRNAs and 53 novel miRNAs targeting genes enriched in biological processes such as floral organ morphogenesis and hormone signaling transudation. We further validated the regulatory relationship between differentially expressed miRNAs and their target genes combining computational prediction, degradome sequencing, and expression pattern analysis. Finally, we integrated weighted gene co-expression analysis and constructed miRNA-mRNA regulatory networks mediating floral bud flushing competency. In general, our study revealed the miRNA-mediated networks in modulating floral bud break in P. mume. The findings will contribute to the comprehensive understanding of miRNA-mediated regulatory mechanism governing floral bud break and dormancy cycling in wood perennials.