Mining Heat-Resistant Key Genes of Peony Based on Weighted Gene Co-Expression Network Analysis.

The RNA-Seq and gene expression data of mature leaves under high temperature stress of Paeonia suffruticosa 'Hu Hong' were used to explore the key genes of heat tolerance of peony. The weighted gene co-expression network analysis (WGCNA) method was used to construct the network, and the main modules and core genes of co-expression were screened according to the results of gene expression and module function enrichment analysis. According to the correlation of gene expression, the network was divided into 19 modules. By analyzing the expression patterns of each module gene, Blue, Salmon and Yellow were identified as the key modules of peony heat response related functions. GO and KEGG functional enrichment analysis was performed on the genes in the three modules and a network diagram was constructed. Based on this, two key genes PsWRKY53 (TRINITY_DN60998_c1_g2, TRINITY_DN71537_c0_g1) and PsHsfB2b (TRINITY_DN56794_c0_g1) were excavated, which may play a key role in the heat shock response of peony. The three co-expression modules and two key genes were helpful to further elucidate the heat resistance mechanism of P. suffruticosa 'Hu Hong'.

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.

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.

Genomic Resequencing Unravels the Genetic Basis of Domestication, Expansion, and Trait Improvement in Morus Atropurpurea.

Mulberry is an economically important plant in the sericulture industry and traditional medicine. However, the genetic and evolutionary history of mulberry remains largely unknown. Here, this work presents the chromosome-level genome assembly of Morus atropurpurea (M. atropurpurea), originating from south China. Population genomic analysis using 425 mulberry accessions reveal that cultivated mulberry is classified into two species, M. atropurpurea and M. alba, which may have originated from two different mulberry progenitors and have independent and parallel domestication in north and south China, respectively. Extensive gene flow is revealed between different mulberry populations, contributing to genetic diversity in modern hybrid cultivars. This work also identifies the genetic architecture of the flowering time and leaf size. In addition, the genomic structure and evolution of sex-determining regions are identified. This study significantly advances the understanding of the genetic basis and domestication history of mulberry in the north and south, and provides valuable molecular markers of desirable traits for mulberry breeding.

Populus D-type cyclin gene PsnCYCD1;1 accelerates cell division and participates in secondary growth of vascular bundles.

Plant growth and development rely heavily on cyclins, which comprise an important class of cell division regulators. D-type cyclins (CYCDs) are responsible for the rate-limiting step of G1 cells. In the plant kingdom, despite the importance of CYCDs in herbaceous plants, there is little knowledge of these proteins in perennial woody plants. Here, the gene of a nucleus-localized cyclin, PsnCYCD1;1, was cloned from Populus simonii × P. nigra. PsnCYCD1;1 was highly expressed in tissues with active cell division, especially the leaf buds, and could be induced by sucrose and phytohormones. Moreover, overexpression of PsnCYCD1;1 in poplar could stimulate cell division, resulting in the generation of small cells and causing severe morphological changes in the vascular bundles, resulting in 'S'-shaped tortuous stems and curled leaves. Furthermore, transcriptomic analysis revealed that endogenous genes related to cell division and vascular cambium development were significantly up-regulated in the transgenic plants. In addition, using yeast two-hybrid and bimolecular fluorescence complementation assays PsnCDKA1, PsnICK3, and PsnICK5 were identified as proteins interacting with PsnCYCD1;1. Our study demonstrates that PsnCYCD1;1 accelerates plant cell division and participates in secondary growth of vascular bundles in poplar.

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.

Integration of Transcriptome and Metabolome Reveals the Formation Mechanism of Red Stem in Prunus mume.

Prunus mume var. purpurea, commonly known as "Red Bone", is a special variety with pink or purple-red xylem. It is famous due to gorgeous petals and delightful aromas, playing important roles in urban landscaping. The regulation mechanism of color formation in P. mume var. purpurea stem development is unclear. Here, we conducted a comprehensive analysis of transcriptome and metabolome in WYY ('Wuyuyu' accession, red stem) and FLE ('Fei Lve' accession, green stem), and found a total of 256 differential metabolites. At least 14 anthocyanins were detected in WYY, wherein cyanidin 3,5-O-diglucoside and peonidin3-O-glucoside were significantly accumulated through LC-MS/MS analysis. Transcriptome data showed that the genes related to flavonoid-anthocyanin biosynthesis pathways were significantly enriched in WYY. The ratio of dihydroflavonol 4-reductase (DFR) and flavonol synthase (FLS) expression levels may affect metabolic balance in WYY, suggesting a vital role in xylem color formation. In addition, several transcription factors were up-regulated, which may be the key factors contributing to transcriptional changes in anthocyanin synthesis. Overall, the results provide a reference for further research on the molecular mechanism of xylem color regulation in P. mume and lay a theoretical foundation for cultivating new varieties.

Ectopic Expression of Poplar PsnCYCD1;1 Reduces Cell Size and Regulates Flower Organ Development in Nicotiana tabacum.

The D-type cyclin (CYCD) gene, as the rate-limiting enzyme in the G1 phase of cell cycle, plays a vital role in the process of plant growth and development. Early studies on plant cyclin mostly focused on herbs, such as Arabidopsis thaliana. The sustainable growth ability of woody plants is a unique characteristic in the study of plant cyclin. Here, the promoter of PsnCYCD1;1 was cloned from poplar by PCR and genetically transformed into tobacco. A strong GUS activity was observed in the areas with vigorous cell division, such as stem tips, lateral buds, and young leaves. The PsnCYCD1;1-GFP fusion expression vector was transformed into tobacco, and the green fluorescence signal was observed in the nucleus. Compared with the control plant, the transgenic tobacco showed significant changes in the flower organs, such as enlargement of sepals, petals, and fruits. Furthermore, the stems of transgenic plants were slightly curved at each stem node, the leaves were curled on the adaxial side, and the fruits were seriously aborted after artificial pollination. Microscopic observation showed that the epidermal cells of petals, leaves, and seed coats of transgenic plants became smaller. The transcriptional levels of endogenous genes, such as NtCYCDs, NtSTM, NtKNAT1, and NtASs, were upregulated by PsnCYCD1;1. Therefore, PsnCYCD1;1 gene played an important role in the regulation of flower organ and stem development, providing new understanding for the functional characterization of CYCD gene and new resources for improving the ornamental value of horticultural plants.

PtaERF194 inhibits plant growth and enhances drought tolerance in poplar.

The water deficits limit the growth and development of agricultural and forest organisms. The AP2/ethylene response factor (ERF) family has been identified as one of the largest plant-specific transcription factors (TFs) essential for plant development and stress response. The function of PtaERF194 in growth and drought tolerance was detected in the overexpression (OX) and RNA interference (RNAi) transgenic poplar 717 hybrids (Populus tremula × Populus alba). Plant growth, stem vessels, water-use efficiency (WUE), chlorophyll content and PtaERF194 co-expressed genes were analyzed using morphological, physiological and molecular methods. Overexpression seedlings showed a shorter and smaller phenotype along with smaller and more vessels compared with the wild-type (WT). Physiological indices indicated that OX with low transpiration and stomatal conductance improved the tolerance to drought by enhancing WUE, limiting water loss and maintaining high water potential. A total of 12 differentially expressed genes co-expressed with PtaERF194 were identified, and they worked together to regulate drought tolerance through the abscisic acid signaling and reactive oxygen species scavenging processes. However, RNAi plants showed similar morphology and physiology to WT, suggesting that the function of PtaERF194 was redundant with other ERF TFs. The findings of the current study may shed new light on the positive function of ERF TFs in plant drought stress tolerance.

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.

Integrative Identification of Crucial Genes Associated With Plant Hormone-Mediated Bud Dormancy in Prunus mume.

Prunus mume is an important ornamental woody plant with winter-flowering property, which is closely related to bud dormancy. Despite recent scientific headway in deciphering the mechanism of bud dormancy in P. mume, the overall picture of gene co-expression regulating P. mume bud dormancy is still unclear. Here a total of 23 modules were screened by weighted gene co-expression network analysis (WGCNA), of which 12 modules were significantly associated with heteroauxin, abscisic acid (ABA), and gibberellin (GA), including GA1, GA3, and GA4. The yellow module, which was positively correlated with the content of ABA and negatively correlated with the content of GA, was composed of 1,426 genes, among which 156 transcription factors (TFs) were annotated with transcriptional regulation function. An enrichment analysis revealed that these genes are related to the dormancy process and plant hormone signal transduction. Interestingly, the expression trends of PmABF2 and PmABF4 genes, the core members of ABA signal transduction, were positively correlated with P. mume bud dormancy. Additionally, the PmSVP gene had attracted lots of attention because of its co-expression, function enrichment, and expression level. PmABF2, PmABF4, and PmSVP were the genes with a high degree of expression in the co-expression network, which was upregulated by ABA treatment. Our results provide insights into the underlying molecular mechanism of plant hormone-regulated dormancy and screen the hub genes involved in bud dormancy in P. mume.

Overexpression Populus d-Type Cyclin Gene PsnCYCD1;1 Influences Cell Division and Produces Curved Leaf in Arabidopsis thaliana.

d-type cyclins (CYCDs) are a special class of cyclins and play extremely important roles in plant growth and development. In the plant kingdom, most of the existing studies on CYCDs have been done on herbaceous plants, with few on perennial woody plants. Here, we identified a Populus d-type cyclin gene, PsnCYCD1;1, which is mainly transcribed in leaf buds and stems. The promoter of PsnCYCD1;1 activated GUS gene expression and transgenic Arabidopsis lines were strongly GUS stained in whole seedlings and mature anthers. Moreover, subcellular localization analysis showed the fluorescence signal of PsnCYCD1;1-GFP fusion protein is present in the nucleus. Furthermore, overexpression of the PsnCYCD1;1 gene in Arabidopsis can promote cell division and lead to small cell generation and cytokinin response, resulting in curved leaves and twisted inflorescence stems. Moreover, the transcriptional levels of endogenous genes, such as ASs, KNATs, EXP10, and PHB, were upregulated by PsnCYCD1;1. Together, our results indicated that PsnCYCD1;1 participates in cell division by cytokinin response, providing new information on controlling plant architecture in woody plants.

Identification of the PmWEEP locus controlling weeping traits in Prunus mume through an integrated genome-wide association study and quantitative trait locus mapping.

Weeping Prunus mume (mei) has long been cultivated in East Asia for its specific ornamental value. However, little is known about the regulatory mechanism of the weeping trait in mei, which limits molecular breeding for the improvement of weeping-type cultivars. Here, we quantified the weeping trait in mei using nested phenotyping of 214 accessions and 342 F1 hybrids. Two major associated loci were identified from the genome-wide association study (GWAS), which was conducted using 3,014,409 single nucleotide polymorphisms (SNPs) derived from resequencing, and 8 QTLs and 55 epistatic loci were identified from QTL mapping using 7,545 specific lengths amplified fragment (SLAF) markers. Notably, an overlapping PmWEEP major QTL was fine mapped within a 0.29 Mb region on chromosome 7 (Pa7), and a core SNP locus closely associated with the weeping trait was screened and validated. Furthermore, a total of 22 genes in the PmWEEP QTL region were expressed in weeping or upright mei based on RNA-seq analysis. Among them, only a novel gene (Pm024213) containing a thioredoxin (Trx) domain was found to be close to the core SNP and specifically expressed in buds and branches of weeping mei. Co-expression analysis of Pm024213 showed that most of the related genes were involved in auxin and lignin biosynthesis. These findings provide insights into the regulatory mechanism of the weeping trait and effective molecular markers for molecular-assisted breeding in Prunus mume.

Research advances in and prospects of ornamental plant genomics.

The term 'ornamental plant' refers to all plants with ornamental value, which generally have beautiful flowers or special plant architectures. China is rich in ornamental plant resources and known as the "mother of gardens". Genomics is the science of studying genomes and is useful for carrying out research on genome evolution, genomic variations, gene regulation, and important biological mechanisms based on detailed genome sequence information. Due to the diversity of ornamental plants and high sequencing costs, the progress of genome research on ornamental plants has been slow for a long time. With the emergence of new sequencing technologies and a reduction in costs since the whole-genome sequencing of the first ornamental plant (Prunus mume) was completed in 2012, whole-genome sequencing of more than 69 ornamental plants has been completed in <10 years. In this review, whole-genome sequencing and resequencing of ornamental plants will be discussed. We provide analysis with regard to basic data from whole-genome studies of important ornamental plants, the regulation of important ornamental traits, and application prospects.

Genome-wide identification, characterisation, and evolution of ABF/AREB subfamily in nine Rosaceae species and expression analysis in mei (Prunus mume).

Rosaceae is an important family containing some of the highly evolved fruit and ornamental plants. Abiotic stress responses play key roles in the seasonal growth and development of plants. However, the molecular basis of stress responses remains largely unknown in Rosaceae. Abscisic acid (ABA) is a stress hormone involving abiotic stress response pathways. The ABRE-binding factor/ABA-responsive element-binding protein (ABF/AREB) is a subfamily of the basic domain/leucine zipper (bZIP) transcription factor family. It plays an important role in the ABA-mediated signaling pathway. Here, we analyzed the ABF/AREB subfamily genes in nine Rosaceae species. A total of 64 ABF/AREB genes were identified, including 18, 28, and 18 genes in the Rosoideae, Amygdaloideae, and Maloideae traditional subfamilies, respectively. The evolutionary relationship of the ABF/AREB subfamily genes was studied through the phylogenetic analysis, the gene structure and conserved motif composition, Ka/Ks values, and interspecies colinearity. These gene sets were clustered into four groups. In the Prunus ABF/AREB (PmABF) promoters, several cis-elements related to light, hormone, and abiotic stress response were predicted. PmABFs expressed in five different tissues, except PmABF5, which expressed only in buds. In the dormancy stages, PmABF1, 2, 5 and 7 showed differential expression. The expression of PmABF3, 4 and 6 was positively correlated with the ABA concentration. Except for PmABF5, all the PmABFs were sensitive to ABA. Several ABRE elements were contained in the promoters of PmABF1, 3, 6, 7. Based on the findings of our study, we speculate that PmABFs may play a role in flower bud dormancy in P. mume.

Comparative gene expression analysis reveals that multiple mechanisms regulate the weeping trait in Prunus mume.

Prunus mume (also known as Mei) is an important ornamental plant that is popular with Asians. The weeping trait in P. mume has attracted the attention of researchers for its high ornamental value. However, the formation of the weeping trait of woody plants is a complex process and the molecular basis of weeping stem development is unclear. Here, the morphological and histochemical characteristics and transcriptome profiles of upright and weeping stems from P. mume were studied. Significant alterations in the histochemical characteristics of upright and weeping stems were observed, and the absence of phloem fibres and less xylem in weeping stems might be responsible for their inability to resist gravity and to grow downward. Transcriptome analysis showed that differentially expressed genes (DEGs) were enriched in phenylpropanoid biosynthesis and phytohormone signal transduction pathways. To investigate the differential responses to hormones, upright and weeping stems were treated with IAA (auxin) and GA3 (gibberellin A3), respectively, and the results revealed that weeping stems had a weaker IAA response ability and reduced upward bending angles than upright stems. On the contrary, weeping stems had increased upward bending angles than upright stems with GA3 treatment. Compared to upright stems, interestingly, DEGs associated with diterpenoid biosynthesis and phenylpropanoid biosynthesis were significantly enriched after being treated with IAA, and expression levels of genes associated with phenylpropanoid biosynthesis, ABC transporters, glycosylphosphatidylinositol (GPI)-anchor biosynthesis were altered after being treated with GA3 in weeping stems. Those results reveal that multiple molecular mechanisms regulate the formation of weeping trait in P. mume, which lays a theoretical foundation for the cultivation of new varieties.