The rhizosphere microbiome and its influence on the accumulation of metabolites in Bletilla striata (Thunb.) Reichb. f.

BACKGROUND: Bletilla striata (Thunb.) Reichb. f. (B. striata) is a perennial herbaceous plant in the Orchidaceae family known for its diverse pharmacological activities, such as promoting wound healing, hemostasis, anti-inflammatory effects, antioxidant properties, and immune regulation. Nevertheless, the microbe-plant-metabolite regulation patterns for B. striata remain largely undetermined, especially in the field of rhizosphere microbes. To elucidate the interrelationships between soil physics and chemistry and rhizosphere microbes and metabolites, a comprehensive approach combining metagenome analysis and targeted metabolomics was employed to investigate the rhizosphere soil and tubers from four provinces and eight production areas in China. RESULTS: Our study reveals that the core rhizosphere microbiome of B. striata is predominantly comprised of Paraburkholderia, Methylibium, Bradyrhizobium, Chitinophaga, and Mycobacterium. These microbial species are recognized as potentially beneficial for plants health. Comprehensive analysis revealed a significant association between the accumulation of metabolites, such as militarine and polysaccharides in B. striata and the composition of rhizosphere microbes at the genus level. Furthermore, we found that the soil environment indirectly influenced the metabolite profile of B. striata by affecting the composition of rhizosphere microbes. Notably, our research identifies soil organic carbon as a primary driving factor influencing metabolite accumulation in B. striata. CONCLUSION: Our fndings contribute to an enhanced understanding of the comprehensive regulatory mechanism involving microbe-plant-metabolite interactions. This research provides a theoretical basis for the cultivation of high-quality traditional Chinese medicine B. striata.

Phosphorylation of 399S at CsHsp70 of Cymbidium sinense is essential to maintain chlorophyll stability.

The Chinese orchids symbolise nobility and gentility in China, and the variation of leaf color makes Cymbidium sinense more diversified and valuable. However, its color variations especially at the protein level still remain largely unexplored. In this study, the proteomics and phosphoproteomics of Cymbidium sinense leaf color variation mutants were studied. A total of 1059 differentially abundant proteins (DAPs) and 1127 differentially abundant phosphorylation sites belonging to 644 phosphoproteins (DAPPs) were identified in the yellow section of leaf variegation mutant of Cymbidium sinense (MY) compared with the green section (MG). Moreover, 349 co-expressing proteins were found in both omics' datasets, while only 26 proteins showed the same expression patterns in the two omics. The interaction network analysis of kinases and phosphatases showed that DAPs and DAPPs in photosynthesis, response to hormones, pigment metabolic process, phosphorylation, glucose metabolic process, and dephosphorylation might contribute to leaf color variation. The abundance of 28 Hsps and 28 phosphorylation sites belonging to 10 Hsps showed significant differences between MG and MY. CsHsp70 was selected to explore the function in Cymbidium sinense leaf variegation. The results showed CsHsp70 is essential for maintaining photosynthetic pigment content and the 399S phosphorylation site is crucial to the function of CsHsp70. Collectively, our findings construct a comprehensive coverage of protein and protein phosphorylation in leaf variegation of C. sinense, providing valuable insights into its formation mechanisms.

Mechanism underlying the rapid growth of Phalaenopsis equestris induced by 60Co-γ-ray irradiation.

Gamma (γ)-ray irradiation is one of the important modern breeding methods. Gamma-ray irradiation can affect the growth rate and other characteristics of plants. Plant growth rate is crucial for plants. In horticultural crops, the growth rate of plants is closely related to the growth of leaves and flowering time, both of which have important ornamental value. In this study, 60Co-γ-ray was used to treat P. equestris plants. After irradiation, the plant's leaf growth rate increased, and sugar content and antioxidant enzyme activity increased. Therefore, we used RNA-seq technology to analyze the differential gene expression and pathways of control leaves and irradiated leaves. Through transcriptome analysis, we investigated the reasons for the rapid growth of P. equestris leaves after irradiation. In the analysis, genes related to cell wall relaxation and glucose metabolism showed differential expression. In addition, the expression level of genes encoding ROS scavenging enzyme synthesis regulatory genes increased after irradiation. We identified two genes related to P. equestris leaf growth using VIGS technology: PeNGA and PeEXPA10. The expression of PeEXPA10, a gene related to cell wall expansion, was down-regulated, cell wall expansion ability decreased, cell size decreased, and leaf growth rate slowed down. The TCP-NGATHA (NGA) molecular regulatory module plays a crucial role in cell proliferation. When the expression of the PeNGA gene decreases, the leaf growth rate increases, and the number of cells increases. After irradiation, PeNGA and PeEXPA10 affect the growth of P. equestris leaves by influencing cell proliferation and cell expansion, respectively. In addition, many genes in the plant hormone signaling pathway show differential expression after irradiation, indicating the crucial role of plant hormones in plant leaf growth. This provides a theoretical basis for future research on leaf development and biological breeding.

Identification and Analysis of PEPC Gene Family Reveals Functional Diversification in Orchidaceae and the Regulation of Bacterial-Type PEPC.

Phosphoenolpyruvate carboxylase (PEPC) gene family plays a crucial role in both plant growth and response to abiotic stress. Approximately half of the Orchidaceae species are estimated to perform CAM pathway, and the availability of sequenced orchid genomes makes them ideal subjects for investigating the PEPC gene family in CAM plants. In this study, a total of 33 PEPC genes were identified across 15 orchids. Specifically, one PEPC gene was found in Cymbidium goeringii and Platanthera guangdongensis; two in Apostasia shenzhenica, Dendrobium chrysotoxum, D. huoshanense, Gastrodia elata, G. menghaiensis, Phalaenopsis aphrodite, Ph. equestris, and Pl. zijinensis; three in C. ensifolium, C. sinense, D. catenatum, D. nobile, and Vanilla planifolia. These PEPC genes were categorized into four subgroups, namely PEPC-i, PEPC-ii, and PEPC-iii (PTPC), and PEPC-iv (BTPC), supported by the comprehensive analyses of their physicochemical properties, motif, and gene structures. Remarkably, PEPC-iv contained a heretofore unreported orchid PEPC gene, identified as VpPEPC4. Differences in the number of PEPC homolog genes among these species were attributed to segmental duplication, whole-genome duplication (WGD), or gene loss events. Cis-elements identified in promoter regions were predominantly associated with light responsiveness, and circadian-related elements were observed in each PEPC-i and PEPC-ii gene. The expression levels of recruited BTPC, VpPEPC4, exhibited a lower expression level than other VpPEPCs in the tested tissues. The expression analyses and RT-qPCR results revealed diverse expression patterns in orchid PEPC genes. Duplicated genes exhibited distinct expression patterns, suggesting functional divergence. This study offered a comprehensive analysis to unveil the evolution and function of PEPC genes in Orchidaceae.

Temperature-Regulated Flowering Locus T-Like Gene Coordinates the Spike Initiation in Phalaenopsis Orchid.

Phalaenopsis aphrodite can be induced to initiate spike growth and flowering by exposure to low ambient temperatures. However, the factors and mechanisms responsible for spike initiation in P. aphrodite remain largely unknown. In this study, we show that a repressor Flowing Locus T-like (FTL) gene, FTL, can act as a negative regulator of spike initiation in P. aphrodite. The mRNA transcripts of PaFTL are consistently high during high ambient temperature, thereby preventing premature spike initiation. However, during low ambient temperature, PaFTL expression falls while FT expression increases, allowing for spike initiation. Knock-down of PaFTL expression through virus-inducing gene silencing promoted spike initiation at 30/28°C. Moreover, PaFTL interacts with FLOWERING LOCUS D in a similar manner to FT to regulate downstream flowering initiation genes. Transgenic P. aphrodite plants exhibiting high expression of PaFTL do not undergo spike initiation, even when exposed to low ambient temperatures. These findings shed light on the flowering mechanisms in Phalaenopsis and provide new insights into how perennial plants govern spike initiation in response to temperature cues.

PeFtsH5 negatively regulates the biological stress response in Phalaenopsis equestris mitochondria.

Mitochondrial homeostasis plays a crucial role in determining cell fate by direct influence on cell apoptosis and autophagy. The ATP and Zn2+-dependent protease FtsH are of paramount importance in maintaining mitochondrial homeostasis. In Phalaenopsis equestris, three mitochondrial FtsH proteases were identified, one of which was encoded by the PeFtsH5 gene. This gene encoded a distinctive mitochondrial protein featuring a unique domain within the FtsH family. Down-regulating the expression of the PeFtsH5 homolog in Nicotiana benthamiana resulted in elevated expression levels of SA synthesis-related genes, leading to enhanced disease resistance. However, this down-regulation also caused cellular damage. Similarly, in P. equestris, the down-regulation of PeFtsH5 expression promoted the expression of defense response genes, leading to accelerated apoptosis and increased ROS levels. Nonetheless, this down-regulation also positively influenced plant resistance to biotic stress. Notably, the PeFtsH5 (i-AAA) protein, as revealed by dual membrane experiments, could form homopolymers exclusively, as it did not interact with the other two mitochondrial FtsH proteases. Consequently, this mitochondrial FtsH protease functioned as a homopolymer within P. equestris cells. The findings of this study elucidated the role of PeFtsH5 in responding to biological stress and provided new insights into its potential molecular mechanism. The result presented in this study hold promise for future research endeavors examining the regulatory effects of mitochondrial proteases on mitochondrial homeostasis and the development of stress-resistant P. equestris varieties through breeding programs.

PeCIN8 expression correlates with flower size and resistance to yellow leaf disease in Phalaenopsis orchids.

BACKGROUND: The orchid industry has seen a recent surge in export values due to the floral morphology and versatile applications of orchids in various markets for medicinal, food additive, and cosmetic usages. However, plant-related diseases, including the yellow leaf disease caused by Fusarium solani, have caused significant losses in the production value of Phalaenopsis (up to 30%). RESULTS: In this study, 203 Phalaenopsis cultivars were collected from 10 local orchid nurseries, and their disease severity index and correlation with flower size were evaluated. Larger flowers had weaker resistance to yellow leaf disease, and smaller flowers had stronger resistance. For the genetic relationship of disease resistance to flower size, the genetic background of all cultivars was assessed using OrchidWiz Orchid Database Software and principal component analysis. In addition, we identified the orthologous genes of BraTCP4, namely PeIN6, PeCIN7, and PeCIN8, which are involved in resistance to pathogens, and analyzed their gene expression. The expression of PeCIN8 was significantly higher in the most resistant cultivars (A7403, A11294, and A2945) relative to the most susceptible cultivars (A10670, A6390, and A10746). CONCLUSIONS: We identified a correlation between flower size and resistance to yellow leaf disease in Phalaenopsis orchids. The expression of PeCIN8 may regulate the two traits in the disease-resistant cultivars. These findings can be applied to Phalaenopsis breeding programs to develop resistant cultivars against yellow leaf disease.

Overexpressing plant ferredoxin-like protein enhances photosynthetic efficiency and carbohydrates accumulation in Phalaenopsis.

Crassulacean acid metabolism (CAM) is one of three major models of carbon dioxide assimilation pathway with better water-use efficiency and slower photosynthetic efficiency in photosynthesis. Previous studies indicated that the gene of sweet pepper plant ferredoxin-like protein (PFLP) shows high homology to the ferredoxin-1(Fd-1) family that belongs to photosynthetic type Fd and involves in photosystem I. It is speculated that overexpressing pflp in the transgenic plant may enhance photosynthetic efficiency through the electron transport chain (ETC). To reveal the function of PFLP in photosynthetic efficiency, pflp transgenic Phalaenopsis, a CAM plant, was generated to analyze photosynthetic markers. Transgenic plants exhibited 1.2-folds of electron transport rate than that of wild type (WT), and higher CO2 assimilation rates up to 1.6 and 1.5-folds samples at 4 pm and 10 pm respectively. Enzyme activity of phosphoenolpyruvate carboxylase (PEPC) was increased to 5.9-folds in Phase III, and NAD+-linked malic enzyme (NAD+-ME) activity increased 1.4-folds in Phase IV in transgenic plants. The photosynthesis products were analyzed between transgenic plants and WT. Soluble sugars contents such as glucose, fructose, and sucrose were found to significantly increase to 1.2, 1.8, and 1.3-folds higher in transgenic plants. The starch grains were also accumulated up to 1.4-folds in transgenic plants than that of WT. These results indicated that overexpressing pflp in transgenic plants increases carbohydrates accumulation by enhancing electron transport flow during photosynthesis. This is the first evidence for the PFLP function in CAM plants. Taken altogether, we suggest that pflp is an applicable gene for agriculture application that enhances electron transport chain efficiency during photosynthesis.

Molecular mechanism of different flower color formation of Cymbidium ensifolium.

Cymbidium ensifolium is one of the national orchids in China, which has high ornamental value with changeable flower colors. To understand the formation mechanism of different flower colors of C. ensifolium, this research conducted transcriptome and metabolome analyses on four different colored sepals of C. ensifolium. Metabolome analysis detected 204 flavonoid metabolites, including 17 polyphenols, 27 anthocyanins, 75 flavones, 34 flavonols, 25 flavonoids, 18 flavanones, and 8 isoflavones. Among them, purple-red and red sepals contain a lot of anthocyanins, including cyanidin, pelargonin, and paeoniflorin, while yellow-green and white sepals have less anthocyanins detected, and their metabolites are mainly flavonols, flavanones and flavonoids. Transcriptome sequencing analysis showed that the expression levels of the anthocyanin biosynthetic enzyme genes in red and purple-red sepals were significantly higher than those in white and yellow-green sepals of C. ensifolium. The experimental results showed that CeF3'H2, CeDFR, CeANS, CeF3H and CeUFGT1 may be the key genes involved in anthocyanin production in C. ensifolium sepals, and CeMYB104 has been proved to play an important role in the flower color formation of C. ensifolium. The results of transformation showed that the CeMYB104 is involved in the synthesis of anthocyanins and can form a purple-red color in the white perianth of Phalaenopsis. These findings provide a theoretical reference to understand the formation mechanism of flower color in C. ensifolium.

Research advances on the gene regulation of floral development and color in orchids.

Orchidaceae is one of the largest monocotyledon families and contributes significantly to worldwide biodiversity, with value in the fields of landscaping, medicine, and ecology. The diverse phenotypes and vibrant colors of orchid floral organs make them excellent research objects for investigating flower development and pigmentation. In recent years, a number of orchid genomes have been published, laying the molecular foundation for revealing flower development and color presentation. In this article, we review transcription factors, the structural genes responsible for the floral pigment synthesis pathways, the molecular mechanisms of flower morphogenesis, and the potential relationship between flower type and flower color. This study provides a theoretical reference for the research on molecular mechanisms related to flower morphogenesis and color presentation, genetic improvement, and new variety creation in orchids.

Comparative Phylogenetic Analysis for Aerides (Aeridinae, Orchidaceae) Based on Six Complete Plastid Genomes.

Aerides Lour. (Orchidaceae, Aeridinae) is a group of epiphytic orchids with high ornamental value, mainly distributed in tropical and subtropical forests, that comprises approximately 20 species. The species are of great value in floriculture and garden designing because of their beautiful flower shapes and colors. Although the morphological boundaries of Aerides are clearly defined, the relationship between Aerides and other closely related genera is still ambiguous in terms of phylogeny. To better understand their phylogenetic relationships, this study used next-generation sequencing technology to investigate the phylogeny and DNA barcoding of this taxonomic unit using genetic information from six Aerides plastid genomes. The quadripartite-structure plastomes ranged from 147,244 bp to 148,391 bp and included 120 genes. Among them, 74 were protein coding genes, 38 were tRNA genes and 8 were rRNA genes, while the ndh genes were pseudogenized or lost. Four non-coding mutational hotspots (rpl20-rpl33, psbM, petB, rpoB-trnCGCA, Pi > 0.06) were identified. A total of 71-77 SSRs and 19-46 long repeats (>30 bp) were recognized in Aerides plastomes, which were mostly located in the large single-copy region. Phylogenetic analysis indicated that Aerides was monophylic and sister to Renanthera. Moreover, our results confirmed that six Aerides species can be divided into three major clades. These findings provide assistance for species identification and DNA barcoding investigation in Aerides, as well as contributes to future research on the phylogenomics of Orchidaceae.

Genome-Wide Identification and Expression Analysis of the SPL Gene Family in Three Orchids.

SPL transcription factors regulate important processes such as plant growth and development, metabolic regulation, and abiotic stress. They play crucial roles in the development of flower organs. However, little is known about the characteristics and functions of the SPLs in the Orchidaceae. In this study, Cymbidium goeringii Rchb. f., Dendrobium chrysotoxum Lindl., and Gastrodia elata BI. were used as research objects. The SPL gene family of these orchids was analyzed on a genome-wide scale, and their physicochemical properties, phylogenetic relationships, gene structures, and expression patterns were studied. Transcriptome and qRT-PCR methods were combined to investigate the regulatory effect of SPLs on the development of flower organs during the flowering process (bud, initial bloom, and full bloom). This study identifies a total of 43 SPLs from C. goeringii (16), D. chrysotoxum (17), and G. elata (10) and divides them into eight subfamilies according to the phylogenetic tree. Most SPL proteins contained conserved SBP domains and complex gene structures; half of the genes had introns longer than 10 kb. The largest number and variety of cis-acting elements associated with light reactions were enriched, accounting for about 45% of the total (444/985); 13/43 SPLs contain response elements of miRNA156. GO enrichment analysis showed that the functions of most SPLs were mainly enriched in the development of plant flower organs and stems. In addition, expression patterns and qRT-PCR analysis suggested the involvement of SPL genes in the regulation of flower organ development in orchids. There was little change in the expression of the CgoSPL in C. goeringii, but DchSPL9 and GelSPL2 showed significant expression during the flowering process of D. chrysotoxum and G. elata, respectively. In summary, this paper provides a reference for exploring the regulation of the SPL gene family in orchids.

Does Reproductive Success in Natural and Anthropogenic Populations of Generalist Epipactis helleborine Depend on Flower Morphology and Nectar Composition?

The purpose of our study was to determine the role of flower structure and nectar composition in shaping the reproductive success (RS) of the generalist orchid Epipactis helleborine in natural and anthropogenic populations. We supposed that the distinct character of two groups of habitats creates different conditions for plant-pollinator relationships, thus influencing reproductive success in E. helleborine populations. Both pollinaria removal (PR) and fruiting (FRS) were differentiated between the populations. On average, FRS was almost two times higher in the anthropogenic than in the natural populations. The difference between the two population groups in PR was smaller but still statistically significant. RS parameters were correlated with some floral display and flower traits. Floral display influenced RS only in three anthropogenic populations. Flower traits had a weak influence on RS (10 of the 192 cases analyzed). The more important trait in shaping RS was nectar chemistry. The nectar of E. helleborine is relatively diluted with a lower sugar concentration in the anthropogenic than in the natural populations. In the natural populations, domination of sucrose over hexoses was found, while in the anthropogenic populations, hexoses were more abundant and the participation of sugars was balanced. In some populations, sugars influenced RS. In E. helleborine nectar, 20 proteogenic and 7 non-proteogenic amino acids (AAs) were found with a clear domination of glutamic acid. We noted relationships between some AAs and RS, but distinct AAs shaped RS in different populations, and their impact was independent of their participation. Our results indicate that the flower structure and nectar composition of E. helleborine reflect its generalistic character and meet the requirements of a wide range of pollinators. Simultaneously, the differentiation of flower traits suggests a variation in pollinator assemblages in particular populations. Knowledge about the factors influencing RS in distinct habitats helps to understand the evolutionary potential of species and to understand mechanisms and processes crucial for shaping interactions between plants and pollinators.

Age-related differences in physiological and metabolic responses of Pleione aurita (Orchidaceae) pseudobulbs to drought stress and recovery.

The pseudobulb is a storage organ for water and nutrients that plays a crucial role in the growth and survival of epiphytic orchids. However, the role of water and metabolites in pseudobulb during adaptation to environmental stress are rarely detected through control experiments. In the present study, water-related physiological traits and metabolite changes in the pseudobulbs at the flowering stage and full leaf expansion stage for Pleione aurita were investigated after drought stress and recovery treatments. We found that the composition of non-structural carbohydrates (starch vs. soluble sugar) varied over the lifetime of pseudobulbs, and older pseudobulbs stored more water, whereas younger pseudobulbs stored more dry matter. When plants were subjected to drought stress and subsequent recovery, multiple metabolites in the pseudobulbs including non-structural carbohydrates, flavonoids, phenolic acids, as well as amino acids and their derivatives responded positively to these water level fluctuations. For those metabolites that differently accumulated in both stress and recovery processes, old pseudobulbs contained a higher number of these key metabolites than did the connected younger pseudobulbs. In addition, young and old pseudobulbs use different metabolic pathways to both respond and recover to drought. These results indicate that orchid pseudobulbs cope with water level fluctuations by mobilizing metabolite reserves and that pseudobulbs of different ages exhibit different physiological and metabolic responses to drought stress. These findings broadens our understanding of the role pseudobulbs play in the survival of orchids growing in epiphytic habitats.

PeGRF6-PeGIF1 complex regulates cell proliferation in the leaf of Phalaenopsis equestris.

Phalaenopsis equestris is an ornamental plant with very large leaves. In this study, we identified genes related to the regulation of leaf development in Phalaenopsis and explored their mechanism of action. Sequence alignment and phylogenetic analyses revealed that PeGRF6 in the PeGRF family of P. equestris has similarities with the Arabidopsis genes AtGRF1 and AtGRF2, which are known to be involved in the regulation of leaf development. Among the PeGRFs, PeGRF6 was continuously and stably expressed at various stages of leaf development. The functions of PeGRF6 and of its complex formed with PeGIF1 in leaf development were verified by virus-induced gene silencing (VIGS) technology. The results show that the PeGRF6-PeGIF1 complex forms in the nucleus and positively regulates leaf cell proliferation via influencing cell size. Interestingly, VIGS suppression of PeGRF6 resulted in anthocyanin accumulation in Phalaenopsis leaves. Analyses of the regulatory mechanism of the miR396-PeGRF6 model based on the P. equestris small RNA library constructed here suggested that PeGRF6 transcripts are cleaved by Peq-miR396. These results show that, compared with PeGRF6 or PeGIF1 alone, the PeGRF6-PeGIF1 complex plays a more important role in the leaf development of Phalaenopsis, possibly by regulating the expression of cell cycle-related genes.

High-quality Cymbidium mannii genome and multifaceted regulation of crassulacean acid metabolism in epiphytes.

Epiphytes with crassulacean acid metabolism (CAM) photosynthesis are widespread among vascular plants, and repeated evolution of CAM photosynthesis is a key innovation for micro-ecosystem adaptation. However, we lack a complete understanding of the molecular regulation of CAM photosynthesis in epiphytes. Here, we report a high-quality chromosome-level genome assembly of a CAM epiphyte, Cymbidium mannii (Orchidaceae). The 2.88-Gb orchid genome with a contig N50 of 22.7 Mb and 27 192 annotated genes was organized into 20 pseudochromosomes, 82.8% of which consisted of repetitive elements. Recent expansions of long terminal repeat retrotransposon families have made a major contribution to the evolution of genome size in Cymbidium orchids. We reveal a holistic scenario of molecular regulation of metabolic physiology using high-resolution transcriptomics, proteomics, and metabolomics data collected across a CAM diel cycle. Patterns of rhythmically oscillating metabolites, especially CAM-related products, reveal circadian rhythmicity in metabolite accumulation in epiphytes. Genome-wide analysis of transcript and protein level regulation revealed phase shifts during the multifaceted regulation of circadian metabolism. Notably, we observed diurnal expression of several core CAM genes (especially ßCA and PPC) that may be involved in temporal fixation of carbon sources. Our study provides a valuable resource for investigating post-transcription and translation scenarios in C. mannii, an Orchidaceae model for understanding the evolution of innovative traits in epiphytes.

Genome-Wide Identification Analysis of the R2R3-MYB Transcription Factor Family in Cymbidium sinense for Insights into Drought Stress Responses.

Cymbidium sinense represents a distinctive Orchidaceae plant that is more tolerant than other terrestrial orchids. Studies have shown that many members of the MYB transcription factor (TF) family, especially the R2R3-MYB subfamily, are responsive to drought stress. This study identified 103 CsMYBs; phylogenetic analysis classified these genes into 22 subgroups with Arabidopsis thaliana. Structural analysis showed that most CsMYB genes contained the same motifs, three exons and two introns, and showed a helix-turn-helix 3D structure in each R repeat. However, the members of subgroup 22 contained only one exon and no intron. Collinear analysis revealed that C. sinense had more orthologous R2R3-MYB genes with wheat than A. thaliana and rice. Ka/Ks ratios indicated that most CsMYB genes were under purifying negative selection pressure. Cis-acting elements analysis revealed that drought-related elements were mainly focused on subgroups 4, 8, 18, 20, 21, and 22, and Mol015419 (S20) contained the most. The transcriptome analysis results showed that expression patterns of most CsMYB genes were upregulated in leaves in response to slight drought stress and downregulated in roots. Among them, members in S8 and S20 significantly responded to drought stress in C. sinense. In addition, S14 and S17 also participated in these responses, and nine genes were selected for the real-time reverse transcription quantitative PCR (RT-qPCR) experiment. The results were roughly consistent with the transcriptome. Our results, thus, provide an important contribution to understanding the role of CsMYBs in stress-related metabolic processes.

Sugars and sucrose transporters in pollinia of Phalaenopsis aphrodite (Orchidaceae).

The pollen grains of Phalaenopsis orchids are clumped tightly together, packed in pollen dispersal units called pollinia. In this study, the morphology, cytology, biochemistry, and sucrose transporters in pollinia of Phalaenopsis orchids were investigated. Histochemical detection was used to characterize the distribution of sugars and callose at the different development stages of pollinia. Ultra-performance liquid chromatography-high resolution-tandem mass spectrometry data indicated that P. aphrodite accumulated abundant saccharides such as sucrose, galactinol, myo-inositol, and glucose, and trace amounts of raffinose and trehalose in mature pollinia. We found that galactinol synthase (PAXXG304680) and trehalose-6-phosphate phosphatase (PAXXG016120) genes were preferentially expressed in mature pollinia. The P. aphrodite genome was identified as having 11 sucrose transporters (SUTs). Our qRT-PCR confirmed that two SUTs (PAXXG030250 and PAXXG195390) were preferentially expressed in the pollinia. Pollinia germinated in pollen germination media (PGM) supplemented with 10% sucrose showed increased callose production and enhanced pollinia germination, but there was no callose or germination in PGM without sucrose. We show that P. aphrodite accumulates high levels of sugars in mature pollinia, providing nutrients and enhanced SUT gene expression for pollinia germination and tube growth.

Two polyamines -responsive WRKY transcription factors from Anoectochilus roxburghii play opposite functions on flower development.

Anoectochilus roxburghii is a rare and precious plant with medicinal and healthcare functions. Embryo abortion caused the lack of resources. Polyamine promoted its flowering and stress resistance in our previous study. But the mechanism remains unclear. The WRKY transcription factor family has been linked to a variety of biological processes in plants. In this study, two WRKY TFs (ArWRKY5 and ArWRKY20) of A. roxburghii, which showed significant response to Spd treatment, were identified and functionally analyzed. Tissue specific expression analyzation showed both of them mostly present in the flower. And ArWRKY5 expressed highest in the flower bud stage (-1 Flowering), while ArWRKY20 showed the highest expression in earlier flower bud stage (-2 Flowering) and the expression gradually decreased with flowering. The transcriptional activation activity assay and subcellular localization revealed that ArWRKY5 and ArWRKY20 were located in the nucleus and ArWRKY20 showed transcriptional activity. The heterologous expression of ArWRKY5 in Arabidopsis thaliana showed earlier flowering, while overexpression of ArWRKY20 delayed flowering. But the OE-ArWRKY20 lines had a robust body shape and a very significant increase in the number of rosette leaves. Furthermore, stamens and seed development were positively regulated by these two ArWRKYs. These results indicated that ArWRKY5 and ArWRKY20 not only play opposite roles in the floral development, but also regulate the plant growth and seed development in A. thaliana. But their specific biological functions and mechanism in A. roxburghii need to be investigated further.

Cloning, subcellular localization and expression analysis of squalene epoxidase gene BsSE1 from Bletilla striata.

Squalene epoxidase catalyzes the oxidation of squalene to 2,3-oxo-squalene (BsSE1), and is the key rate limiting enzyme in the synthesis of triterpenoids and sterols in plants. This study focused on the basic aspects of BsSE1 including the sequence information, sub-cellular localization expression patterns of BsSE1. Using to the sequence information of Bletilla striata transcriptome, the full-length CDS of BsSE1 gene was amplified. The physicochemical properties and structural characteristics of BsSE1 protein were analyzed by bioinformatics analysis software, and vector was constructed to analyze the protein locations and expression patterns. The results showed that the CDS of BsSE1 gene was 1542 bp, encoding 513 amino acids. BsSE1 protein is a hydrophobic protein with two transmembrane domains but no signal peptides. It is localied in the endoplasmic reticulum membrane and belongs to the typical squalene epoxidase gene. BsSE1 has the closest genetic relationship with SE protein of Dendrobium officinale and Phalaenopsis equestris. The expression level of BsSE1 was higher in pseudobulblet of Bletilla striata seedlings, followed by roots, and lower in seedling stems. After SA induction, the expression of BsSE1 in Bletilla striata showed significant changes, increased first, then decreased, finally increase again. The results provide a basis for further study of this gene family in plants.