Genome-wide identification and characterization of TCP gene family in Dendrobium nobile and their role in perianth development.

TCP is a widely distributed, essential plant transcription factor that regulates plant growth and development. An in-depth study of TCP genes in Dendrobium nobile, a crucial parent in genetic breeding and an excellent model material to explore perianth development in Dendrobium, has not been conducted. We identified 23 DnTCP genes unevenly distributed across 19 chromosomes and classified them as Class I PCF (12 members), Class II: CIN (10 members), and CYC/TB1 (1 member) based on the conserved domain and phylogenetic analysis. Most DnTCPs in the same subclade had similar gene and motif structures. Segmental duplication was the predominant duplication event for TCP genes, and no tandem duplication was observed. Seven genes in the CIN subclade had potential miR319 and -159 target sites. Cis-acting element analysis showed that most DnTCP genes contained many developmental stress-, light-, and phytohormone-responsive elements in their promoter regions. Distinct expression patterns were observed among the 23 DnTCP genes, suggesting that these genes have diverse regulatory roles at different stages of perianth development or in different organs. For instance, DnTCP4 and DnTCP18 play a role in early perianth development, and DnTCP5 and DnTCP10 are significantly expressed during late perianth development. DnTCP17, 20, 21, and 22 are the most likely to be involved in perianth and leaf development. DnTCP11 was significantly expressed in the gynandrium. Specially, MADS-specific binding sites were present in most DnTCP genes putative promoters, and two Class I DnTCPs were in the nucleus and interacted with each other or with the MADS-box. The interactions between TCP and the MADS-box have been described for the first time in orchids, which broadens our understanding of the regulatory network of TCP involved in perianth development in orchids.

Isolation of Tulasnella spp. from Cultivated Paphiopedilum Orchids and Screening of Germination-Enhancing Fungi.

Ex situ conservation, an important way to increase the survival and sustainability of endangered species, is widely used in the conservation of endangered orchids. However, long-term ex situ conservation might affect the dominant group of orchid symbiotic fungi, which are crucial for orchid growth and reintroduction. This study investigated the culturable Tulasnella spp. associated with Paphiopedilum orchids after long-term greenhouse cultivation, and identified germination-enhancing isolates. A total of 44 Tulasnella isolates were obtained from the roots of 14 Paphiopedilum spp., and 29 of them were selected for phylogenetic analysis. They clustered mainly with Tulasnella deliquescens, Tulasnella calospora, Tulasnella bifrons, and Tulasnella irregularis, but included two potential new groups. Compared with published uncultured data, most of the isolates were grouped together with the reported types, and the dominant Tulasnella associated with P. armeniacum and P. micranthum could still be isolated after ten years of cultivation, most of which were the first isolation. In vitro symbiotic germination showed that certain root isolates could promote seed germination (e.g., parm152 isolated from P. armeniacum, Php12 from P. hirsutissimum, and prhi68 from P. rhizomatosum). These data indicated that the dominant Tulasnella types colonizing the roots of cultivated Paphiopedilum are stable over time, and germination-enhancing fungi colonizing the roots would benefit for seed reproduction after population reintroduction into the wild.

New Insights into the Mechanism of Spatiotemporal Scent Accumulation in Orchid Flowers.

Orchid flowers have a unique structure that consists of three sepals and three petals, with one of the petals forming the labellum (lip) that can be differentiated into the hypochile and epichile. In orchids, the emission of floral scent is specific and spatially complex. Little is understood about the molecular and biochemical mechanisms of the differing scent emissions between the parts of orchid flowers. Here, we investigated this in the Cattleya hybrid KOVA, and our study showed that monoterpenes, including linalool and geraniol, are the main components responsible for the KOVA floral scent. The KOVA flower was scentless to the human nose before it reached full bloom, potentially because the 1-deoxy-d-xylulose 5-phosphate synthases (RcDXSs) and 4-hydroxy-3-methylbut-2-enyl diphosphate synthases (RcHDSs) that biosynthesize monoterpenes were highly expressed in flowers only when it reached full flowering. Additionally, the spatial expression profile of the monoterpene synthases (RcMTPSs), which were highly expressed in the basal region of the lip (hypochile), contributed to the highest monoterpene emissions from this part of the flower. This might have caused the hypochile to be more fragrant than the other parts of the flower. These findings enrich our understanding of the difference in scents between different flower parts in plants and provide information to breed novel orchid cultivars with special floral scents.

Reducing Nitrogen Rate and Increasing Plant Density Accomplished High Yields with Satisfied Grain Quality of Soft Wheat via Modifying the Free Amino Acid Supply and Storage Protein Gene Expression.

In a 2 yr field experiment, we investigated the combined effects of reduced nitrogen (N) rate and increased plant density on the trade-off between the grain protein content (GPC) and the grain yield (GY) in soft wheat cultivars. Reducing N application significantly decreased both GPC and GY; however, to some extent, increasing the top-dressed N ratio and plant density compensated for the GY loss. Optimizing the combination of these three factors (150 kg N ha-1 with 50% top-dressed N and 360 × 104 plants ha-1) achieved both the required lower GPC for soft wheat and relatively higher GY compared with the conventional cultivation strategy. In addition, this optimized combination downregulated 11 high-molecular-weight glutenin subunits, 8 low-molecular-weight glutenin subunits, 5 α/ß-gliadins, and 2 γ-gliadins in mature grains as identified by data-independent acquisition mass spectrometry. Further analysis indicated that the relatively lower free amino acid content and downregulated expressions of the seed storage protein (SSP) synthesis-related genes in filling grains contributed to the reduction of SSP and GPC. Furthermore, the dilution effect induced by a relatively higher accumulation of starch than proteins also partially explained the reduced GPC. Unlike proteins, grain starch accumulation and content depended more on the soluble sugar availability, rather than on the starch synthesis capacity. These findings provide novel insights on simultaneous improvement in the grain quality and yield of soft wheat through synchronized manipulations of N fertilization and plant density.

New insight into the molecular mechanism of colour differentiation among floral segments in orchids.

An unbalanced pigment distribution among the sepal and petal segments results in various colour patterns of orchid flowers. Here, we explored this type of mechanism of colour pattern formation in flowers of the Cattleya hybrid 'KOVA'. Our study showed that pigment accumulation displayed obvious spatiotemporal specificity in the flowers and was likely regulated by three R2R3-MYB transcription factors. Before flowering, RcPAP1 was specifically expressed in the epichile to activate the anthocyanin biosynthesis pathway, which caused substantial cyanin accumulation and resulted in a purple-red colour. After flowering, the expression of RcPAP2 resulted in a low level of cyanin accumulation in the perianths and a pale pink colour, whereas RcPCP1 was expressed only in the hypochile, where it promoted α-carotene and lutein accumulation and resulted in a yellow colour. Additionally, we propose that the spatiotemporal expression of different combinations of AP3- and AGL6-like genes might participate in KOVA flower colour pattern formation.

Correction: Wan et al. Circadian Regulation of Alternative Splicing of Drought-Associated CIPK Genes in Dendrobium catenatum (Orchidaceae). Int. J. Mol. Sci. 2019, 20, 688.

We would like to submit several corrections to our published paper "Circadian Regulation of Alternative Splicing of Drought-Associated CIPK Genes in Dendrobium catenatum (Orchidaceae)"  [...].

Circadian Regulation of Alternative Splicing of Drought-Associated CIPK Genes in Dendrobium catenatum (Orchidaceae).

Dendrobium catenatum, an epiphytic and lithophytic species, suffers frequently from perennial shortage of water in the wild. The molecular mechanisms of this orchid's tolerance to abiotic stress, especially drought, remain largely unknown. It is well-known that CBL-interacting protein kinase (CIPKs) proteins play important roles in plant developmental processes, signal transduction, and responses to abiotic stress. To study the CIPKs' functions for D. catenatum, we first identified 24 CIPK genes from it. We divided them into three subgroups, with varying intron numbers and protein motifs, based on phylogeny analysis. Expression patterns of CIPK family genes in different tissues and in response to either drought or cold stresses suggested DcaCIPK11 may be associated with signal transduction and energy metabolism. DcaCIPK9, -14, and -16 are predicted to play critical roles during drought treatment specifically. Furthermore, transcript expression abundances of DcaCIPK16 showed polar opposites during day and night. Whether under drought treatment or not, DcaCIPK16 tended to emphatically express transcript1 during the day and transcript3 at night. This implied that expression of the transcripts might be regulated by circadian rhythm. qRT-PCR analysis also indicated that DcaCIPK3, -8, and -20 were strongly influenced by circadian rhythmicity. In contrast with previous studies, for the first time to our knowledge, our study revealed that the major CIPK gene transcript expressed was not always the same and was affected by the biological clock, providing a different perspective on alternative splicing preference.

RNA-seq transcriptomic profiling of crassulacean acid metabolism pathway in Dendrobium catenatum.

The regulation of crassulacean acid metabolism (CAM) pathway has recently become a topic of intensive research and has been explored in terms of several aspects, including phylogenetics, genomics, and transcriptomics. Orchidaceae, which contains approximately 9,000 CAM species, is one of the largest lineages using this special photosynthetic pathway. However, no comprehensive transcriptomic profiling focused on CAM regulation in orchid species had previously been performed. In this report, we present two Illumina RNA-seq datasets, including a total of 24 mature leaf samples with 844.4 million reads, from Dendrobium catenatum (Orchidaceae), a facultative CAM species. The first dataset was generated from a time-course experiment based on the typical CAM phases in a diel. The second was derived from an experiment on drought stress and stress removal. A series of quality assessments were conducted to verify the reliability of the datasets. These transcriptomic profiling datasets will be useful to explore and understand the essence of CAM regulation.

Transcriptomic profiling for prolonged drought in Dendrobium catenatum.

Orchid epiphytes, a group containing at least 18,000 species, thrive in habitats that often undergo periodic drought stress. However, few global gene expression profiling datasets have been published for studies addressing the drought-resistant mechanism of this special population. In this study, an experiment involving the effect of continuous drought treatments on an epiphytic orchid, Dendrobium catenatum, was designed to generate 39 mature-leaf-tissue RNA-seq sequencing datasets with over two billion reads. These datasets were validated by a series of quality assessments including RNA sample quality, RNA-seq read quality, and global gene expression profiling. We believe that these comprehensive transcriptomic resources will allow a better understanding of the drought-resistant mechanisms of orchid epiphytes.

Photosynthetic, morphological, and reproductive variations in Cypripedium tibeticum in relation to different light regimes in a subalpine forest.

Cypripedium tibeticum, a subalpine orchid species, inhabits various habitats of subalpine forests, mainly including the forest edge (FE), forest gap (FG), and understory (UST), which have significantly different light intensities (FE > FG > UST). However, the ecological and physiological influences caused by different light regimes in this species are still poorly understood. In the present study, photosynthetic, morphological, and reproductive characteristics were comprehensively studied in plants of C. tibeticum grown in three types of habitats. The photosynthetic capacities, such as the net photosynthetic rate, light-saturated photosynthesis (Pmax), and dry mass per unit leaf area (LMA), were higher in FE and FG than in UST according to light availability. Compared with FG, the populations in FE and UST suffer from excessively strong and inadequate radiation, respectively, which was further corroborated by the low Fv/Fm in FE and high apparent quantum yield (AQY) in FG. The leaves of the orchids had various proportions of constituents, such as the leaf area, thickness and (or) epidermal hair, to reduce damage from high radiation (including ultraviolet-b radiation) in FE and capture more light in FG and UST. Although the flower rate (FR) was positively correlated to both Pmax and the daily mean PAR, fruit-set only occurred in the populations in FG. The failures in FE and UST might be ascribed to changes in the floral functional structure and low biomass accumulation, respectively. Moreover, analysis of the demographic statistics showed that FG was an advantageous habitat for the orchid. Thus, C. tibeticum reacted to photosynthetic and morphological changes to adapt to different subalpine forest habitats, and neither full (under FE) nor low (UST) illumination was favorable for population expansion. These findings could serve as a guide for the protection and reintroduction of C. tibeticum and emphasize the importance of specific habitats for Cypripedium spp.

Evolutionary history of PEPC genes in green plants: Implications for the evolution of CAM in orchids.

The phosphoenolpyruvate carboxylase (PEPC) gene is the key enzyme in CAM and C4 photosynthesis. A detailed phylogenetic analysis of the PEPC family was performed using sequences from 60 available published plant genomes, the Phalaenopsis equestris genome and RNA-Seq of 15 additional orchid species. The PEPC family consists of three distinct subfamilies, PPC-1, PPC-2, and PPC-3, all of which share a recent common ancestor in chlorophyte algae. The eudicot PPC-1 lineage separated into two clades due to whole genome duplication (WGD). Similarly, the monocot PPC-1 lineage also divided into PPC-1M1 and PPC-1M2 through an ancient duplication event. The monocot CAM- or C4-related PEPC originated from the clade PPC-1M1. WGD may not be the major driver for the performance of CAM function by PEPC, although it increased the number of copies of the PEPC gene. CAM may have evolved early in monocots, as the CAM-related PEPC of orchids originated from the monocot ancient duplication, and the earliest CAM-related PEPC may have evolved immediately after the diversification of monocots, with CAM developing prior to C4. Our results represent the most complete evolutionary history of PEPC genes in green plants to date and particularly elucidate the origin of PEPC in orchids.

Transcriptomic Analysis of Paeonia delavayi Wild Population Flowers to Identify Differentially Expressed Genes Involved in Purple-Red and Yellow Petal Pigmentation.

Tree peony (Paeonia suffruticosa Andrews) is a very famous traditional ornamental plant in China. P. delavayi is a species endemic to Southwest China that has aroused great interest from researchers as a precious genetic resource for flower color breeding. However, the current understanding of the molecular mechanisms of flower pigmentation in this plant is limited, hindering the genetic engineering of novel flower color in tree peonies. In this study, we conducted a large-scale transcriptome analysis based on Illumina HiSeq sequencing of cDNA libraries generated from yellow and purple-red P. delavayi petals. A total of 90,202 unigenes were obtained by de novo assembly, with an average length of 721 nt. Using Blastx, 44,811 unigenes (49.68%) were found to have significant similarity to accessions in the NR, NT, and Swiss-Prot databases. We also examined COG, GO and KEGG annotations to better understand the functions of these unigenes. Further analysis of the two digital transcriptomes revealed that 6,855 unigenes were differentially expressed between yellow and purple-red flower petals, with 3,430 up-regulated and 3,425 down-regulated. According to the RNA-Seq data and qRT-PCR analysis, we proposed that four up-regulated key structural genes, including F3H, DFR, ANS and 3GT, might play an important role in purple-red petal pigmentation, while high co-expression of THC2'GT, CHI and FNS II ensures the accumulation of pigments contributing to the yellow color. We also found 50 differentially expressed transcription factors that might be involved in flavonoid biosynthesis. This study is the first to report genetic information for P. delavayi. The large number of gene sequences produced by transcriptome sequencing and the candidate genes identified using pathway mapping and expression profiles will provide a valuable resource for future association studies aimed at better understanding the molecular mechanisms underlying flower pigmentation in tree peonies.