Establishment of a Homologous Silencing System with Intact-Plant Infiltration and Minimized Operation for Studying Gene Function in Herbaceous Peonies.

Gene function verification is a crucial step in studying the molecular mechanisms regulating various plant life activities. However, a stable and efficient homologous genetic transgenic system for herbaceous peonies has not been established. In this study, using virus-induced gene silencing technology (VIGS), a highly efficient homologous transient verification system with distinctive advantages was proposed, which not only achieves true "intact-plant" infiltration but also minimizes the operation. One-year-old roots of the representative species, Paeonia lactiflora Pall., were used as the materials; prechilling (4 °C) treatment for 3-5 weeks was applied as a critical precondition for P. lactiflora to acquire a certain chilling accumulation. A dormancy-related gene named HOMEOBOX PROTEIN 31 (PlHB31), believed to negatively regulate bud endodormancy release (BER), was chosen as the target gene in this study. GFP fluorescence was detected in directly infiltrated and newly developed roots and buds; the transgenic plantlets exhibited remarkably earlier budbreak, and PlHB31 was significantly downregulated in silenced plantlets. This study established a homologous transient silencing system featuring intact-plant infiltration and minimized manipulation for gene function research, and also offers technical support and serves as a theoretical basis for gene function discovery in numerous other geophytes.

Genome and whole-genome resequencing of Cinnamomum camphora elucidate its dominance in subtropical urban landscapes.

BACKGROUND: Lauraceae is well known for its significant phylogenetic position as well as important economic and ornamental value; however, most evergreen species in Lauraceae are restricted to tropical regions. In contrast, camphor tree (Cinnamomum camphora) is the most dominant evergreen broadleaved tree in subtropical urban landscapes. RESULTS: Here, we present a high-quality reference genome of C. camphora and conduct comparative genomics between C. camphora and C. kanehirae. Our findings demonstrated the significance of key genes in circadian rhythms and phenylpropanoid metabolism in enhancing cold response, and terpene synthases (TPSs) improved defence response with tandem duplication and gene cluster formation in C. camphora. Additionally, the first comprehensive catalogue of C. camphora based on whole-genome resequencing of 75 accessions was constructed, which confirmed the crucial roles of the above pathways and revealed candidate genes under selection in more popular C. camphora, and indicated that enhancing environmental adaptation is the primary force driving C. camphora breeding and dominance. CONCLUSIONS: These results decipher the dominance of C. camphora in subtropical urban landscapes and provide abundant genomic resources for enlarging the application scopes of evergreen broadleaved trees.

Development of a Multi-Criteria Decision-Making Approach for Evaluating the Comprehensive Application of Herbaceous Peony at Low Latitudes.

The growing region of herbaceous peony (Paeonia lactiflora) has been severely constrained due to the intensification of global warming and extreme weather events, especially at low latitudes. Assessing and selecting stress-tolerant and high-quality peony germplasm is essential for maintaining the normal growth and application of peonies under adverse conditions. This study proposed a modified multi-criteria decision-making (MCDM) model for assessing peonies adapted to low-latitude climates based on our previous study. This model is low-cost, timesaving and suitable for screening the adapted peony germplasm under hot and humid climates. The evaluation was conducted through the analytic hierarchy process (AHP), three major criteria, including adaptability-related, ornamental feature-related and growth habits-related criteria, and eighteen sub-criteria were proposed and constructed in this study. The model was validated on fifteen herbaceous peonies cultivars from different latitudes. The results showed that 'Meiju', 'Hang Baishao', 'Hongpan Tuojin' and 'Bo Baishao' were assessed as Level I, which have strong growth adaptability and high ornamental values, and were recommended for promotion and application at low latitudes. The reliability and stability of the MCDM model were further confirmed by measuring the chlorophyll fluorescence of the selected adaptive cultivars 'Meiju' and 'Hang Baishao' and one maladaptive cultivar 'Zhuguang'. This study could provide a reference for the introduction, breeding and application of perennials under everchanging unfavorable climatic conditions.

Later Growth Cessation and Increased Freezing Tolerance Potentially Result in Later Dormancy in Evergreen Iris Compared with Deciduous Iris.

Winter dormancy is a protective survival strategy for plants to resist harsh natural environments. In the context of global warming, the progression of dormancy has been significantly affected in perennials, which requires further research. Here, a systematic study was performed to compare the induction of dormancy in two closely related iris species with an ecodormancy-only process, the evergreen Iris japonica Thunb. and the deciduous Iris tectorum Maxim. under artificial conditions. Firstly, morphological and physiological observations were evaluated to ensure the developmental status of the two iris species. Furthermore, the expression patterns of the genes involved in key pathways related to plant winter dormancy were determined, and correlation analyses with dormancy marker genes were conducted. We found that deciduous iris entered dormancy earlier than evergreen iris under artificial dormancy induction conditions. Phytohormones and carbohydrates play roles in coordinating growth and stress responses during dormancy induction in both iris species. Moreover, dormancy-related MADS-box genes and SnRKs (Snf1-related protein kinase) might represent a bridge between carbohydrate and phytohormone interaction during iris dormancy. These findings provide a hypothetical model explaining the later dormancy in evergreen iris compared with deciduous iris under artificial dormancy induction conditions and reveal some candidate genes. The findings of this study could provide new insights into the research of dormancy in perennial plants with an ecodormancy-only process and contribute to effectively managing iris production, postharvest storage, and shipping.

Integrative Comparative Assessment of Cold Acclimation in Evergreen and Deciduous Iris Species.

Cold acclimation (CA) is a strategy which plants have evolved to increase freezing tolerance. Global climate change could obstruct CA and raise the probability of winter injury, especially for evergreens. Hence, understanding the regulatory mechanism of CA is crucial to improve freezing tolerance in evergreen plants. A comparative study on a pair of closely related evergreen and deciduous iris species in response to cold through CA was conducive to uncovering and complementing the knowledge of CA. We investigated morphological, physiological and biochemical changes, as well as the expression of associated genes in the functional leaves of both iris species from natural CA to deacclimation. Briefly, fast and strong CA in the evergreen iris might cause early expressions of BAM1, NCED3, GPX6, etc., which leads to strong enzyme activity of starch degradation, abscisic acid biosynthesis and reactive oxygen species scavenging. Additionally, genes belonging to the antioxidant system were mainly induced during deacclimation. These results suggest that interspecies differences in the leaf freezing tolerance of irises are associated with the rate and degree of CA, which activates multiple signaling networks with complex interactions and induces the transcription of cold-responsive genes. Moreover, the ICE-CBF-COR signaling cascade may integrate and initiate diverse cold-responsive pathways during CA of the evergreen iris. The findings of this study provide valuable insight to further research on CA mechanisms and implicate genes which could support breeding strategies in herbaceous perennials under climate changes.

Hybrid RNA Sequencing Strategy for the Dynamic Transcriptomes of Winter Dormancy in an Evergreen Herbaceous Perennial, Iris japonica.

Japanese iris (Iris japonica) is a popular perennial ornamental that originated in China; it has a long display period and remains green outdoors throughout the year. winter dormancy characteristics contribute greatly to the evergreenness of herbaceous perennials. Thus, it is crucial to explore the mechanism of winter dormancy in this evergreen herbaceous perennial. Here, we used the hybrid RNA-seq strategy including single-molecule real-time (SMRT) and next-generation sequencing (NGS) technologies to generate large-scale Full-length transcripts to examine the shoot apical meristems of Japanese iris. A total of 10.57 Gb clean data for SMRT and over 142 Gb clean data for NGS were generated. Using hybrid error correction, 58,654 full-length transcripts were acquired and comprehensively analysed, and their expression levels were validated by real-time qPCR. This is the first full-length RNA-seq study in the Iris genus; our results provide a valuable resource and improve understanding of RNA processing in this genus, for which little genomic information is available as yet. In addition, our data will facilitate in-depth analyses of winter dormancy mechanisms in herbaceous perennials, especially evergreen monocotyledons.

MADS-box transcription factors determine the duration of temporary winter dormancy in closely related evergreen and deciduous Iris spp.

Winter dormancy (WD) is a crucial strategy for plants coping with potentially deadly environments. In recent decades, this process has been extensively studied in economically important perennial eudicots due to changing climate. However, in evergreen monocots with no chilling requirements, dormancy processes are so far a mystery. In this study, we compared the WD process in closely related evergreen (Iris japonica) and deciduous (I. tectorum) iris species across crucial developmental time points. Both iris species exhibit a 'temporary' WD process with distinct durations, and could easily resume growth under warm conditions. To decipher transcriptional changes, full-length sequencing for evergreen iris and short read RNA sequencing for deciduous iris were applied to generate respective reference transcriptomes. Combining results from a multipronged approach, SHORT VEGETATIVE PHASE and FRUITFULL (FUL) from MADS-box was associated with a dormancy- and a growth-related module, respectively. They were co-expressed with genes involved in phytohormone signaling, carbohydrate metabolism, and environmental adaptation. Also, gene expression patterns and physiological changes in the above pathways highlighted potential abscisic acid and jasmonic acid antagonism in coordinating growth and stress responses, whereas differences in carbohydrate metabolism and reactive oxygen species scavenging might lead to species-specific WD durations. Moreover, a detailed analysis of MIKCCMADS-box in irises revealed common features described in eudicots as well as possible new roles for monocots during temporary WD, such as FLOWERING LOCUS C and FUL. In essence, our results not only provide a portrait of temporary WD in perennial monocots but also offer new insights into the regulatory mechanism underlying WD in plants.

Chilling Requirement Validation and Physiological and Molecular Responses of the Bud Endodormancy Release in Paeonia lactiflora 'Meiju'.

The introduction of herbaceous peony (Paeonia lactiflora Pall.) in low-latitude areas is of great significance to expand the landscape application of this world-famous ornamental. With the hazards of climate warming, warm winters occurs frequently, which makes many excellent northern herbaceous peony cultivars unable to meet their chilling requirements (CR) and leads to their poor growth and flowering in southern China. Exploring the endodormancy release mechanism of underground buds is crucial for improving low-CR cultivar screening and breeding. A systematic study was conducted on P. lactiflora 'Meiju', a screened cultivar with a typical low-CR trait introduced from northern China, at the morphological, physiological and molecular levels. The CR value of 'Meiju' was further verified as 677.5 CUs based on the UT model and morphological observation. As a kind of signal transducer, reactive oxygen species (ROS) released a signal to enter dormancy, which led to corresponding changes in carbohydrate and hormone metabolism in buds, thus promoting underground buds to acquire strong cold resistance and enter endodormancy. The expression of important genes related to ABA metabolism, such as NCED3, PP2C, CBF4 and ABF2, reached peaks at the critical stage of endodormancy release (9 January) and then decreased rapidly; the expression of the GA2ox8 gene related to GA synthesis increased significantly in the early stage of endodormancy release and decreased rapidly after the release of ecodormancy (23 January). Cytological observation showed that the period when the sugar and starch contents decreased and the ABA/GA ratio decreased was when 'Meiju' bud endodormancy was released. This study reveals the endodormancy regulation mechanism of 'Meiju' buds with the low-CR trait, which lays a theoretical foundation for breeding new herbaceous peony cultivars with the low-CR trait.

Comparative Study on Physiological Responses and Gene Expression of Bud Endodormancy Release Between Two Herbaceous Peony Cultivars (Paeonia lactiflora Pall.) With Contrasting Chilling Requirements.

With the global temperature increase, diverse endogenous factors and environmental cues can lead to severe obstacles to bud endodormancy release for important economic plants, such as herbaceous peony (Paeonia lactiflora Pall.). Knowing the underlying mechanism in bud endodormancy release is vital for widely planting herbaceous peony at low latitudes with warm winter climates. A systematic study was carried out between the southern Chinese cultivar 'Hang Baishao' with low-chilling requirement (CR) trait and the northern cultivar 'Zhuguang' with high-CR trait. Peony buds were sampled at regular intervals under natural cold during the crucial bud endodormancy release stage. Physiology and morphology of the buds were observed, and the roles of reactive oxygen species (ROS) and relevant genes in the regulation of bud endodormancy release were also highlighted, which has been rather rare in previous bud dormancy studies of both herbaceous and tree peonies. The expression of the starch metabolism- and sucrose synthesis-related genes PlAMY PlSPS and PlSUS was lower in the high-CR 'Zhuguang' and corresponded to a lower content of soluble sugars. The expression of polyamine oxidase gene PlPAO2 correlated with a higher level of hydrogen peroxide (H2O2) in high-CR 'Zhuguang' than in low CR 'Hang Baishao' during bud endodormancy. Expression of PlMAPKKK5, an intermediate gene in the abscisic acid (ABA) response to ROS signaling, correlated with ROS levels and ABA content. We present the hypothesis that accumulation of ROS increases ABA content and decreases GA3 content and signal transduction leading to reduced expression of PlSVP and PlSOC1. Reduced cell division and increased cellular damage which probably blocked bud endodormancy release were also observed in high-CR 'Zhuguang' through histological observation and related genes expression. This study provides a comparative analysis on physiological responses and gene expression patterns of bud dormancy of geophytes in an increasingly unsuitable environment.