Integrated analysis of miRNAome transcriptome and degradome reveals miRNA-target modules governing floral florescence development and senescence across early- and late-flowering genotypes in tree peony

As a candidate national flower of China, tree peony has extremely high ornamental, medicinal and oil value. However, the short florescence and rarity of early-flowering and late-flowering varieties restrict further improvement of the economic value of tree peony. Specific miRNAs and their target genes engaged in tree peony floral florescence, development and senescence remain unknown. This report presents the integrated analysis of the miRNAome, transcriptome and degradome of tree peony petals collected from blooming, initial flowering, full blooming and decay stages in early-flowering variety Paeonia ostii ‘Fengdan’, an early-flowering mutant line of Paeonia ostii ‘Fengdan’ and late-flowering variety Paeonia suffruticosa ‘Lianhe’. Transcriptome analysis revealed a transcript (‘psu.G.00014095’) which was annotated as a xyloglucan endotransglycosylase/hydrolase precursor XTH-25 and found to be differentially expressed across flower developmental stages in Paeonia ostii ‘Fengdan’ and Paeonia suffruticosa ‘Lianhe’. The miRNA-mRNA modules were presented significant enrichment in various pathways such as plant hormone signal transduction, indole alkaloid biosynthesis, arachidonic acid metabolism, folate biosynthesis, fatty acid elongation, and the MAPK signaling pathway. Multiple miRNA-mRNA-TF modules demonstrated the potential functions of MYB-related, bHLH, Trihelix, NAC, GRAS and HD-ZIP TF families in floral florescence, development, and senescence of tree peony. Comparative spatio-temporal expression investigation of eight floral-favored miRNA-target modules suggested that transcript ‘psu.T.00024044’ and microRNA mtr-miR166g-5p are involved in the floral florescence, development and senescence associated agronomic traits of tree peony. The results might accelerate the understanding of the potential regulation mechanism in regards to floral florescence, development and abscission, and supply guidance for tree peony breeding of varieties with later and longer florescence characteristics

Structure and phylogenetic analysis of Paeonia lactiflora ‘Lv He’ chloroplast genome

Economic value development of peony is limited by the fuzzy genetic relationships. Phylogeny investigation, genomic resource conservation, and genome evolution could be elucidated by the chloroplast genome (cp) on account of the extremely conservative structure. Paeonia lactiflora ‘Lv He’, a new peony variety blooming with green flowers, possess excellent ornamental value. Molecular structure and phylogenetic analysis of the plastome of Paeonia lactiflora ‘Lv He’ was resolved in this study. The complete cp genome contained a typical quadripartite structure with a length of 152, 702 bp and an overall 38.4% GC content. The circular molecule of the genome was constituted of a large single-copy (LSC) region of 84,373 bp with 36.7% GC content, a small single-copy (SSC) region of 16,969 bp with 32.7% GC content, and a pair of inverted repeat (IRa and IRb) regions of 25,680 bp each with 43.1% GC content. The genome annotated 115 genes, containg 81 protein-coding genes, 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. Phylogenetic analysis revealed a sister taxonomical relationship to P. lactiflora and P. obovate

Acorn germination and seedling survival of

• We studied the effects of acorn cotyledon loss by insect damage and artificial excision on germination and seedling survival of Quercus variabilis, to determine whether insect infestation decreases acorn germination and seedling establishment; and to what extent cotyledon damage can affect acorn germination and seedling establishment. • Artificial excision partially imitated the effect of insect damage on acorn germination and seedling survival, and indicated that insect damage did not significantly decrease germination rate and seedling survival of Q. variabilis acorns. The viability of infested but still viable acorns strongly depends on which sector weevil larvae feed on. • The cotyledon reserves at the apex of acorns were more important than those at the base in supporting acorn viability and seedling establishment. The excision of apical cotyledon has the most detrimental effects on acorn germination activity. The relatively low effect of larval infestation on acorn viability was likely related to the high frequency of feeding and escaping at the base, which was closely correlated with differentiation in pericarp thickness in Q. variabilis acorns. These could explain the low decrease in acorn germination and seedling survival experienced by infested acorns

Foraging Behavior and Pollination Efficiency of <i>Apis mellifera</i> L. on the Oil Tree Peony ‘Feng Dan’ (<i>Paeonia ostii</i> T. Hong et J.X. Zhang)

To solve the issue of insufficient pollinating of insects for the oil tree peony &#8216;Feng Dan&#8217; (Paeonia ostii T. Hong et J.X. Zhang) and improve its seed set and yield, we conducted observations from 2017 to 2018 to investigate the relationship between honey bee (Apis mellifera L.) foraging behavior and diurnal activity. We compared the single-fruit seed set ratio among three flower types on the same plants of the oil tree peony, which flowered simultaneously, in three pollination areas (bee pollination, natural field pollination, and controlled pollination by pollinators) and in a net room under self-pollination, wind pollination and bee pollination. Apis mellifera exhibited short single visitations, long visitations to a single flower and repeated visits to flowers of the oil tree peony. The number of flower visits of A. mellifera was significantly and positively yet weakly correlated with the number of stigma visits (2017: r = 0.045, p &lt; 0.05; 2018: r = 0.195, p &lt; 0.01). The seed set of oil tree peony follicles in the A. mellifera pollination area was significantly higher than that in the natural pollination field area and the control net rooms. On the same oil tree peony plant with synchronous flowering, the percent seed set of follicles pollinated by A. mellifera at a high density was significantly higher than that resulting from wind pollination and self-pollination

Phylogeny analysis of a wild species of Paeonia lactiflora from Henan province based on the complete chloroplast genome

The complete chloroplast genome sequence of a wild Paeonia lactiflora species from Henan province, Paeonia obovata subsp. Willmottiae (Stapf) D. Y. Hong et K. Y. Pan, was applied to support the difference of Paeonia lactiflora by next-generation sequencing. The length of complete chloroplast genome is 152,660 bp, including a large single-copy (LSC) region, a small single-copy (SSC) region, and a pair of identical inverted repeat (IR) regions (IRa and IRb) with length of 84,363 bp, 16,983 bp and 25,657 bp, respectively. Altogether, 115 genes in chloroplast genome were annotated including 81 protein-encoding genes, 30 tRNA genes, and 4 rRNA genes. Paeonia obovata subsp. Willmottiae (Stapf) D. Y. Hong et K. Y. Pan clustered in the clade of Paeonia lactiflora showed a closer relationship with Paeonia sect. Mudan rather than Paeonia Brownii

Identification of differentially expressed miRNAs and their target genes in response to brassinolide treatment on flowering of tree peony (Paeonia ostii)

Tree peony is a famous flower plant in China, but the short and concentrated flowering period limits its ornamental value and economic value. Brassinolide (BR) plays an important role in plant growth and development including flowering. There have been a large number of reports on the molecular aspects of the flowering process, but the genetic mechanism that was responsible for miRNA-guided regulation of tree peony is almost unclear. In this study, the leaves of tree peony cultivar, ‘Feng Dan’, were sprayed with different concentrations of BR, and the obvious bloom delay was found at the treatment with BR 50 μg/L. The small RNA sequencing and transcriptome sequencing were performed on the petals of tree peony under an untreated control (CK) and the treatment with BR 50 μg/L during four consecutive flowering development stages. A total of 22 known miRNAs belonging to 12 families were identified and 84 novel miRNAs were predicted. Combined with transcriptome data, a total of 376 target genes were predicted for the 18 differentially expressed known miRNAs and 177 target genes were predicted for the 23 differentially expressed novel miRNAs. Additionally, the potential miRNAs and their target genes were identified, including miR156b targeting SPL, miR172a_4 targeting AP2 and four novel miRNAs targeting SPA1, and revealed that they might affect the flowering time in tree peony. Collectively, these results would provide a theoretical basis for further analysis of miRNA-guided regulation on flowering period in tree peony

Molecular mechanism of somatic embryogenesis in paeonia ostii ‘Fengdan’ based on transcriptome analysis combined histomorphological observation and metabolite determination

Abstract Background Tree peony (Paeonia sect. Moutan DC.) is a famous flower native to China with high ornamental, medicinal, and oil value. However, the low regeneration rate of callus is one of the main constraints for the establishment of a genetic transformation system in tree peony. By histomorphological observation, transcriptomic analysis and metabolite determination, we investigated the molecular mechanism of somatic embryogenesis after the establishment of a culture system and the induction of somatic embryo(SE) formation. Results We found that SE formation was successfully induced when cotyledons were used as explants. A total of 3185 differentially expressed genes were screened by comparative transcriptomic analysis of embryogenic callus (EC), SE, and non-embryogenic callus (NEC). Compared to NEC, the auxin synthesis-related genes GH3.6 and PCO2 were up-regulated, whereas cytokinin dehydrogenase (CKX6) and CYP450 family genes were down-regulated in somatic embryogenesis. In SE, the auxin content was significantly higher than the cytokinin content. The methyltransferase-related gene S-adenosylmethionine synthase (SAMS) and the flavonoid biosynthesis-related gene (ANS and F3’5’H) were down-regulated in somatic embryogenesis. The determination of flavonoids showed that rhoifolin and hyperoside had the highest content in SE. The results of transcriptome analysis were consistent with the relative expression of 8 candidate genes by quantitative polymerase chain reaction analysis. Conclusion The results revealed that auxin and cytokinin may play a key role in ‘Fengdan’ somatic embryogenesis. The genes related to somatic embryogenesis were revealed, which has partly elucidated the molecular mechanism of somatic embryogenesis in ‘Fengdan’

Effects of leaf zeatin and zeatin riboside induced by different clipping heights on the regrowth capacity of ryegrass

The effect of clipping height on ryegrass regrowth was investigated by examining the roles of several plant hormones. Our study consisted of three treatment conditions: (1) darkness over whole plants, (2) darkness only over stubble leaf sheaths, and (3) light over whole plants. Results showed that under darkness over whole plant, low stubble height resulted in low leaf regrowth biomass. Similar leaf regrowth biomass was observed under conditions of darkness only over stubble leaf sheaths as well as light over whole plants. Each unit weight of stubble at different clipping heights has relatively similar potential of providing stored organic substance for leaf regrowth. Therefore, regrowth index, calculated as newly grown leaf biomass divided by unit stubble weight, was used to evaluate regrowth capacity at different clipping heights under minimal influence of organic substances stored in stubbles. Under light over whole plants and single clipping, low stubble height and high stubble height with root thinning resulted in low leaf biomass and high regrowth index. On the other hand, under light over whole plants and frequent clipping high leaf biomass and regrowth index were observed in high stubble height. In addition, we found that leaf zeatin and zeatin riboside (Z + ZR) affected ryegrass regrowth and that roots regulated leaf Z + ZR concentration. Thus, our results indicate that root-derived cytokinin concentration in leaves influences ryegrass regrowth at different clipping heights

Characterization of PsmiR319 during flower development in early- and late-flowering tree peonies cultivars

The flowering period is the most important ornamental trait of tree peony, while industrial development of tree peony has been limited by short flowering period. miR319 plays an important regulatory role in plant flowering. In the current study, the expression characteristics and evolution of PsmiR319 in tree peony flowering was explored using ‘Feng Dan’ and ‘Lian He’, which are early-flowering and late-flowering varieties of tree peony, respectively. The structure, evolution, and target(s) of PsmiR319 were analyzed by bioinformatics. Evolution analysis showed that pre-PsmiR319 was distributed in 41 plant species, among which the length of the precursor sequence exhibited marked differences (between 52 and 308 bp). Pre-PsmiR319 of tree peony was located close to the corresponding sequences of Linum usitatissimum and Picea abies in the phylogenetic tree, and in addition, could form a typical hairpin structure including a mature body with a length of 20 bp located on the 3p arm and part of the loop sequence. The mature sequence of miR319 was highly conserved among different species. Target genes of PsmiR319 include MYB-related transcription factor in tree peony. Expression of PsmiR319, assayed by qRT-PCR, differed between ‘Feng Dan’ and ‘Lian He’ during different flower development periods. PsmiR319 and its target gene showed a negative expression regulation relationship during the periods of CE (color exposure), BS (blooming stage), IF (initial flowering), and HO (half opening) in the early-flowering ‘Feng Dan’, and the same in FB (Full blooming) periods of late-flowering ‘Lian He’. Findings from this study provide a reference for further investigation into the mechanism of miR319 in the development of different varieties of tree peony

Screening of Genes Related to Early and Late Flowering in Tree Peony Based on Bulked Segregant RNA Sequencing and Verification by Quantitative Real-Time PCR

Tree peony (Paeonia suffruticosa Andrews) is a perennial woody shrub bearing large and colorful flowers. However, the flowering period is short and relatively uniform, which to an important extent hinders the cultivation and exploitation of ornamental peonies. In this study, the segregation of an F1 population derived from P. ostti ‘Feng Dan’ (an early-flowering cultivar) × P. suffruticosa ‘Xin Riyuejin’ (a late-flowering cultivar) was used to screen and analyze candidate genes associated with flowering period of the two parents. Extreme early- and late-flowering genotypes of the F1 population at full-bloom stage were sampled to establish an early-flowering mixed pool (T03), a late-flowering mixed pool (T04), a late-flowering male pool (T01), and an early-flowering female pool (T02), using the Sequencing By Synthesis (SBS) technology on the Illumina HiSeq TM2500 platform. A total of 56.51 Gb of clean reads data, comprising at least 87.62% of Quality30 (Q30), was generated, which was then combined into 173,960 transcripts (N50 = 1781) and 78,645 (N50 = 1282) unigenes, with a mean length of 1106.76 and 732.27 base pairs (bp), respectively. Altogether, 58,084 genes were annotated by comparison with public databases, based on an E-value parameter of less than 10−5 and 10−10 for BLAST and HMMER, respectively. In total, 291 unigene sequences were finally screened out by BSR-seq (bulked segregant RNA-seq) association analysis. To validate these unigenes, we finally confirmed seven unigenes that were related to early and late flowering, which were then verified by quantitative real-time PCR (qRT-PCR). This is the first reported study to screen genes associated with early and late flowering of tree peony by the BSA (bulked sample analysis) method of transcriptome sequencing, and to construct a high-quality transcriptome database. A set of candidate functional genes related to flowering time was successfully obtained, providing an important genetic resource for further studies of flowering in peony and the mechanism of regulation of flowering time in tree peony