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.

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.

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.

Cypripedium macranthos Swartz, C. × ventricosum Swartz, and C. shanxiense S. C. Chen were highly promising ornamental plants. In this study, the latest complete chloroplast genome sequence of C. macranthos, C. × ventricosum, and C. shanxiense were reported using correct sample material from their native range, and their phylogenetic relationships with other related species were investigated preliminarily. The whole chloroplast genome lengths of C. macranthos, C. × ventricosum, and C. shanxiense were 181,030 bp, 175,385 bp, and 177,627 bp, respectively, with total GC contents of 34.56%, 34.48%, and 34.42%, respectively. Based on the maximum likelihood phylogenetic tree from the chloroplast genome sequences and internal transcribed spacer (ITS) sequences, it was confirmed that C. × ventricosum is most closely related to C. calceolus, and the idea that C. × ventricosum is an interspecific hybrid between C. calceolus and C. macranthos is supported. By integrating the results of phylogenetic analysis, genomic structural comparison, and considerations of sampling locations, it is evident that the former chloroplast genome of C. macranthos is inaccurate. This study provides crucial information for research on the phylogeny, genetics, and conservation of C. macranthos, C. × ventricosum, and C. shanxiense.

Aluminum-activated malate transporter (ALMT) genes play an important role in aluminum ion (Al³⁺) tolerance, fruit acidity, and stomatal movement. Although decades of research have been carried out in many plants, there is little knowledge about the roles of ALMT in Orchidaceae. In this study, 34 ALMT genes were identified in the genomes of four orchid species. Specifically, ten ALMT genes were found in Dendrobium chrysotoxum and D. catenatum, and seven were found in Apostasia shenzhenica and Phalaenopsis equestris. These ALMT genes were further categorized into four clades (clades 1–4) based on phylogenetic relationships. Sequence alignment and conserved motif analysis revealed that most orchid ALMT proteins contain conserved regions (TM1, GABA binding motif, and WEP motif). We also discovered a unique motif (19) belonging to clade 1, which can serve as a specifically identified characteristic. Comparison with the gene structure of AtALMT genes (Arabidopsis thaliana) showed that the gene structure of ALMT was conserved across species, but the introns were longer in orchids. The promoters of orchid ALMT genes contain many light-responsive and hormone-responsive elements, suggesting that their expression may be regulated by light and phytohormones. Chromosomal localization and collinear analysis of D. chrysotoxum indicated that tandem duplication (TD) is the main reason for the difference in the number of ALMT genes in these orchids. D. catenatum was chosen for the RT-qPCR experiment, and the results showed that the DcaALMT gene expression pattern varied in different tissues. The expression of DcaALMT1-9 was significantly changed after ABA treatment. Combining the circadian CO₂ uptake rate, titratable total acid, and RT-qPCR data analysis, most DcaALMT genes were highly expressed at night and around dawn. The result revealed that DcaALMT genes might be involved in photosynthate accumulation. The above study provides more comprehensive information for the ALMT gene family in Orchidaceae and a basis for subsequent functional analysis.

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)”