NAC transcription factors (TFs) participate in multiple biological processes, including biotic and abiotic stress responses, signal transduction and development. Cold stress can adversely impact plant growth and development, thereby limiting agricultural productivity. Prunus mume, an excellent horticultural crop, is widely cultivated in Asian countries. Its flower can tolerate freezing-stress in the early spring. To investigate the putative NAC genes responsible for cold-stress, we identified and analyzed 113 high-confidence PmNAC genes and characterized them by bioinformatics tools and expression profiles. These PmNACs were clustered into 14 sub-families and distributed on eight chromosomes and scaffolds, with the highest number located on chromosome 3. Duplicated events resulted in a large gene family; 15 and 8 pairs of PmNACs were the result of tandem and segmental duplicates, respectively. Moreover, three membrane-bound proteins (PmNAC59/66/73) and three miRNA-targeted genes (PmNAC40/41/83) were identified. Most PmNAC genes presented tissue-specific and time-specific expression patterns. Sixteen PmNACs (PmNAC11/19/20/23/41/48/58/74/75/76/78/79/85/86/103/111) exhibited down-regulation during flower bud opening and are, therefore, putative candidates for dormancy and cold-tolerance. Seventeen genes (PmNAC11/12/17/21/29/42/30/48/59/66/73/75/85/86/93/99/111) were highly expressed in stem during winter and are putative candidates for freezing resistance. The cold-stress response pattern of 15 putative PmNACs was observed under 4 °C at different treatment times. The expression of 10 genes (PmNAC11/20/23/40/42/48/57/60/66/86) was upregulated, while 5 genes (PmNAC59/61/82/85/107) were significantly inhibited. The putative candidates, thus identified, have the potential for breeding the cold-tolerant horticultural plants. This study increases our understanding of functions of the NAC gene family in cold tolerance, thereby potentially intensifying the molecular breeding programs of woody plants.

Summary: The Orchidaceae is of economic and ecological importance and constitutes ˜10% of all seed plant species. Here, we report a genome physical map forCymbidium sinense, a well‐known species belonging to genusCymbidiumthat has thousands of natural variation varieties of flower organs, flower and leaf colours and also referred as the King of Fragrance, which make it arose into a unique cultural symbol in China. The high‐quality chromosome‐scale genome assembly was 3.52 Gb in size, 29 638 protein‐coding genes were predicted, and evidence for whole‐genome duplication shared with other orchids was provided. Marked amplification of cytochrome‐ and photosystem‐related genes was observed, which was consistent with the shade tolerance and dark green leaves ofC. sinense. Extensive duplication of MADS‐box genes, and the resulting subfunctional and expressional differentiation, was associated with regulation of species‐specific flower traits, including wild‐type and mutant‐type floral patterning, seasonal flowering and ecological adaption.CsSEP4was originally found to positively regulate gynostemium development. TheCsSVPgenes and their interaction proteins CsAP1 and CsSOC1 were significantly expanded and involved in the regulation of low‐temperature‐dependent flowering. Important genetic clues to the colourful leaf traits, purple‐black flowers and volatile trait inC.sinensewere also found. The results provide new insights into the molecular mechanisms of important phenotypic traits ofCymbidiumand its evolution and serve as a powerful platform for future evolutionary studies and molecular breeding of orchids.

The colorful leaf is an important ornamental character of Cymbidium sinense (C. sinense), especially the red leaf, which has always been attracted by breeders and consumers. However, little is documented on the formation mechanism of the red leaf of C. sinense. In this study, the changing patterns of flavonoid-related metabolites, corresponding enzyme activities and genes expression in the leaves of C. sinense ‘Red Sun’ from red to yellow and finally to green was investigated. A total of 196 flavonoid-related metabolites including 11 anthocyanins metabolites were identified using UPLC-MS/MS-based approach. In the process of leaf color change, 42 metabolites were identified as having significantly different contents and the content of 28 differential metabolites turned to zero. In anthocyanin biosynthetic pathway, content of all 15 identified metabolites showed downregulation trend in the process of leaf color change. Among the 15 metabolites, the contents of Naringenin chalcone, Pelargonidin O-acetylhexoside and Anthocyanin 3-O-beta-d-glucoside decreased to zero in the green leaf stage. The changing pattern of enzyme activity of 10 enzymes involved in the anthocyanin biosynthetic pathway showed different trends from red leaves that have turned yellow and finally green, while the expression of genes encoding these enzymes was all down-regulated in the process of leaf color change. The results of this study revealed the types of flavonoid-related metabolites and the comprehensive analysis of metabolites content, enzyme activities and genes expression providing a new reference for breeders to improve the leaf color of C. sinense ‘Red Sun’.

Versatile protoplast platforms greatly facilitate the development of modern botany. However, efficient protoplast-based systems are still challenging for numerous horticultural plants and crops. Orchids are globally cultivated ornamental and medicinal monocot plants, but few efficient protoplast isolation and transient expression systems have been developed. In this study, we established a highly efficient orchid protoplast isolation protocol by selecting suitable source materials and optimizing the enzymatic conditions, which required optimal D-mannitol concentrations (0.4–0.6 M) combined with optimal 1.2% cellulose and 0.6% macerozyme, 5 μM of 2-mercaptoethanol and 6 h digestion. Tissue- and organ-specific protoplasts were successfully isolated from young leaves [∼3.22 × 10⁶/g fresh weight (FW)], flower pedicels (∼5.26 × 10⁶/g FW), and young root tips (∼7.66 × 10⁵/g FW) of Cymbidium orchids. This protocol recommends the leaf base tissues (the tender part of young leaves attached to the stem) as better source materials. High yielding viable protoplasts were isolated from the leaf base of Cymbidium (∼2.50 × 10⁷/g FW), Phalaenopsis (1.83 × 10⁷/g FW), Paphiopedilum (1.10 × 10⁷/g FW), Dendrobium (8.21 × 10⁶/g FW), Arundina (3.78 × 10⁶/g FW) orchids, and other economically important monocot crops including maize (Zea mays) (3.25 × 10⁷/g FW) and rice (Oryza sativa) (4.31 × 10⁷/g FW), which showed marked advantages over previous mesophyll protoplast isolation protocols. Leaf base protoplasts of Cymbidium orchids were used for polyethylene glycol (PEG)-mediated transfection, and a transfection efficiency of more than 80% was achieved. This leaf base protoplast system was applied successfully to analyze the CsDELLA-mediated gibberellin signaling in Cymbidium orchids. We investigated the subcellular localization of the CsDELLA-green fluorescent protein fusion and analyzed the role of CsDELLA in the regulation of gibberellin to flowering-related genes via efficient transient overexpression and gene silencing of CsDELLA in Cymbidium protoplasts. This protoplast isolation and transient expression system is the most efficient based on the documented results to date. It can be widely used for cellular and molecular studies in orchids and other economically important monocot crops, especially for those lacking an efficient genetic transformation system in vivo.

Plants facing the seasonal variations always need a growth restraining mechanism when temperatures turn down. C-repeat binding factor (CBF) genes work essentially in the cold perception. Despite lots of researches on CBFs, the multiple crosstalk is still interesting on their interaction with hormones and dormancy-associated MADS (DAM) genes in the growth and dormancy control. Therefore, this study highlights roles of PmCBFs in cold-induced dormancy from different orgens. And a sense-response relationship between PmCBFs and PmDAMs is exhibited in this process, jointly regulated by six PmCBFs and PmDAM4–6. Meantime, GA3 and ABA showed negative and positive correlation with PmCBFs expression levels, respectively. We also find a high correlation between IAA and PmDAM1–3. Finally, we display the interaction mode of PmCBFs and PmDAMs, especially PmCBF1-PmDAM1. These results can disclose another view of molecular mechanism in plant growth between cold-response pathway and dormancy regulation together with genes and hormones.