Selection and Validation of qRT-PCR Internal Reference Genes to Study Flower Color Formation in Camellia impressinervis.

Real-time quantitative PCR (qRT-PCR) is a pivotal technique for gene expression analysis. To ensure reliable and accurate results, the internal reference genes must exhibit stable expression across varied experimental conditions. Currently, no internal reference genes for Camellia impressinervis have been established. This study aimed to identify stable internal reference genes from eight candidates derived from different developmental stages of C. impressinervis flowers. We employed geNorm, NormFinder, and BestKeeper to evaluate the expression stability of these candidates, which was followed by a comprehensive stability analysis. The results indicated that CiTUB, a tubulin gene, exhibited the most stable expression among the eight reference gene candidates in the petals. Subsequently, CiTUB was utilized as an internal reference for the qRT-PCR analysis of six genes implicated in the petal pigment synthesis pathway of C. impressinervis. The qRT-PCR results were corroborated by transcriptome sequencing data, affirming the stability and suitability of CiTUB as a reference gene. This study marks the first identification of stable internal reference genes within the entire genome of C. impressinervis, establishing a foundation for future gene expression and functional studies. Identifying such stable reference genes is crucial for advancing molecular research on C. impressinervis.

Identification of two Bacillus thuringiensis Cry3Aa toxin-binding aminopeptidase N from Rhynchophorus ferrugineus (Coleoptera: Curculionidae).

Rhynchophorus ferrugineus is a quarantine pest that mainly damages plants in tropical regions, which are essential economic resources. Cry3Aa has been used to control coleopteran pests and is known to be toxic to R. ferrugineus. The binding of the Cry toxin to specific receptors on the target insect plays a crucial role in the toxicological mechanism of Cry toxins. However, in the case of R. ferrugineus, the nature and identity of the receptor proteins involved remain unknown. In the present study, pull-down assays and mass spectrometry were used to identify two proteins of aminopeptidase N proteins (RfAPN2a and RfAPN2b) in the larval midguts of R. ferrugineus. Cry3Aa was able to bind to RfAPN2a (Kd = 108.5 nM) and RfAPN2b (Kd = 68.2 nM), as well as midgut brush border membrane vesicles (Kd = 482.5 nM). In silico analysis of both RfAPN proteins included the signal peptide and anchored sites for glycosyl phosphatidyl inositol. In addition, RfAPN2a and RfAPN2b were expressed in the human embryonic kidney 293T cell line, and cytotoxicity assays showed that the transgenic cells were not susceptible to activated Cry3Aa. Our results show that RfAPN2a and RfAPN2b are Cry3Aa-binding proteins involved in the Cry3Aa toxicity of R. ferrugineus. This study deepens our understanding of the action mechanism of Cry3Aa in R. ferrugineus larvae.

The Euscaphis japonica genome and the evolution of malvids.

Malvids is one of the largest clades of rosids, includes 58 families and exhibits remarkable morphological and ecological diversity. Here, we report a high-quality chromosome-level genome assembly for Euscaphis japonica, an early-diverging species within malvids. Genome-based phylogenetic analysis suggests that the unstable phylogenetic position of E. japonica may result from incomplete lineage sorting and hybridization event during the diversification of the ancestral population of malvids. Euscaphis japonica experienced two polyploidization events: the ancient whole genome triplication event shared with most eudicots (commonly known as the γ event) and a more recent whole genome duplication event, unique to E. japonica. By resequencing 101 samples from 11 populations, we speculate that the temperature has led to the differentiation of the evergreen and deciduous of E. japonica and the completely different population histories of these two groups. In total, 1012 candidate positively selected genes in the evergreen were detected, some of which are involved in flower and fruit development. We found that reddening and dehiscence of the E. japonica pericarp and long fruit-hanging time promoted the reproduction of E. japonica populations, and revealed the expression patterns of genes related to fruit reddening, dehiscence and abscission. The key genes involved in pentacyclic triterpene synthesis in E. japonica were identified, and different expression patterns of these genes may contribute to pentacyclic triterpene diversification. Our work sheds light on the evolution of E. japonica and malvids, particularly on the diversification of E. japonica and the genetic basis for their fruit dehiscence and abscission.

Genome-wide identification, evolution and expression analysis of the aspartic protease gene family during rapid growth of moso bamboo (Phyllostachys edulis) shoots.

BACKGROUND: Aspartic proteases (APs) are a class of aspartic peptidases belonging to nine proteolytic enzyme families whose members are widely distributed in biological organisms. APs play essential functions during plant development and environmental adaptation. However, there are few reports about APs in fast-growing moso bamboo. RESULT: In this study, we identified a total of 129 AP proteins (PhAPs) encoded by the moso bamboo genome. Phylogenetic and gene structure analyses showed that these 129 PhAPs could be divided into three categories (categories A, B and C). The PhAP gene family in moso bamboo may have undergone gene expansion, especially the members of categories A and B, although homologs of some members in category C have been lost. The chromosomal location of PhAPs suggested that segmental and tandem duplication events were critical for PhAP gene expansion. Promoter analysis revealed that PhAPs in moso bamboo may be involved in plant development and responses to environmental stress. Furthermore, PhAPs showed tissue-specific expression patterns and may play important roles in rapid growth, including programmed cell death, cell division and elongation, by integrating environmental signals such as light and gibberellin signals. CONCLUSION: Comprehensive analysis of the AP gene family in moso bamboo suggests that PhAPs have experienced gene expansion that is distinct from that in rice and may play an important role in moso bamboo organ development and rapid growth. Our results provide a direction and lay a foundation for further analysis of plant AP genes to clarify their function during rapid growth.

[A new lignan from Euscaphis konishii].

The chemical constituents from the extract of the twigs of Euscaphis konishii with anti-hepatoma activity were investigated, twelve compounds by repeated chromatography with silica gel, Sephadex LH-20 and preparative-HPLC. The structures of the chemical components were elucidated by spectroscopy methods, as konilignan(1),(7R, 8S)-dihydrodehydrodico-niferylalcohol-9-O-ß-D-glucopyranoside(2),illiciumlignan B(3),threo-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-1,3-panediol(4),erythro-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-1,3-panediol(5), matairesinol(6), wikstromol(7), isolariciresinol(8),(+)-lyoniresinol(9), 4-ketopinoresinol(10), syringaresin(11), and vladinol D(12). Among them, compound 1 is a new lignan. Compounds 10 and 12 had moderate inhibitory activity on HepG2 cells, with IC_(50) values of 107.12 µmol·L~(-1) and 183.56 µmol·L~(-1), respectively.

[Chemical constituents from the leaves of Cinnamomum camphora var. linaloolifera and their anti-inflammatory activities].

Thirteen compounds were isolated and purified from the leaves of Cinnamomum camphora by the macroporous resin,silica gel,and Sephadex LH-20 column chromatographies. Those compounds were further identified by IR,UV,MS,and NMR techniques:( 2 S)-1-( 3″,4″-methylenedioxy phenyl)-3-( 2',6'-dimethoxy-4'-hydroxyphenyl)-propan-2-ol( 1),( 2 R,3 R)-5,7-dimethoxy-3',4'-methylenedioxy flavanol( 2),9-hydroxysesamin( 3),sesamin( 4),piperitol( 5),kobusin( 6),(-)-aptosimon( 7),acuminatolide( 8),1ß,11-dihydroxy-5-eudesmene( 9),lasiodiplodin( 10),vanillin( 11),p-hydroxybenzaldehyde( 12),and p-hydroxybenzoic acid ethyl ester( 13). Compound 1 was a novel compound,and compounds 2,6,7,9 and 10 were isolated from Cinnamomum plants for the first time. Compounds 4,7 and 10 were found to possess good inhibitory effect on IL-6 production in LPS-induced BV2 cells at a concentration of 20 µmol·L-1 in the in vitro bioassay,with inhibition rates of 51. 26% ± 4. 13%,67. 82% ± 3. 77% and85. 81%±1. 19%,respectively.

Characterization of Bacterial Communities Associated with Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) and its Host Phoenix sylvestris.

This study measured the changes of microorganisms in the midgut and habitat niche of Rhynchophorus ferrugineus Olivier, an invasive quarantine pest, by Illumina sequencing. The bacterial diversity in the R. ferrugineus larvae midgut and their habitat niche was compared to the uninfected P. sylvestris. The Proteobacteria and Firmicutes occupied a dominant position in the R. ferrugineus midgut and infected P. sylvestris, while in the uninfected P. sylvestris the predominant bacterial phylum was the Cyanobacteria. Enterobacter, Dysgonomonas, and Entomoplasma were the dominant bacterial genera in R. ferrugineus midgut and also within the infected trees and uninfected trees with low relative abundance. These bacteria could be exploited as the biopesticide vector to control R. ferrugineus population. Besides, Sphingobacterium, Shinella, and Rhodobacter genera had the same distribution pattern in the infected and uninfected P. sylvestris, and these bacteria were not found in the midgut of R. ferrugineus. Interestingly, Paludibacter and Parabacteroides were only distributed in the wood fiber of the infected P. sylvestris, which could be used as potential microbial markers to detect if the palm plants are damaged by the R. ferrugineus. The results of this study will be beneficial to the development of control strategies for R. ferrugineus.

Comparative transcriptome among Euscaphis konishii Hayata tissues and analysis of genes involved in flavonoid biosynthesis and accumulation.

BACHGROUND: Euscaphis konishii Hayata, a member of the Staphyleaceae Family, is a plant that has been widely used in Traditional Chinese Medicine and it has been the source for several types of flavonoids. To identify candidate genes involved in flavonoid biosynthesis and accumulation, we analyzed transcriptome data from three E. konishii tissues (leaf, branch and capsule) using Illumina Hiseq 2000 platform. RESULTS: A total of 91.7, 100.3 and 100.1million clean reads were acquired for the leaf, branch and capsule, respectively; and 85,342 unigenes with a mean length of 893.60 bp and N50 length of 1307 nt were assembled using Trinity program. BLASTx analysis allowed to annotate 40,218 unigenes using public protein databases, including NR, KOG/COG/eggNOG, Swiss-Prot, KEGG and GO. A total of 14,291 (16.75%) unigenes were assigned to 128 KEGG pathways, and 900 unigenes were annotated into 22 KEGG secondary metabolites, including flavonoid biosynthesis. The structure enzymes involved in flavonoid biosynthesis, such as phenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate CoA ligase, shikimate O-hydroxycinnamoyltransferase, coumaroylquinate 3'-monooxygenase, caffeoyl-CoA O-methyltransferase, chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase, flavonolsynthese, dihydroflavonol 4-reductase, anthocyanidinreductase, leucoanthocyanidin dioxygenase, leucoanthocyanidin reductase, were identified in the transcriptome data, 40 UDP-glycosyltransferase (UGT), 122 Cytochrome P450 (CYP) and 25 O-methyltransferase (OMT) unigenes were also found. A total of 295 unigenes involved in flavonoid transport and 220 transcription factors (97 MYB, 84 bHLH and 39 WD40) were identified. Furthermore, their expression patterns among different tissues were analyzed by DESeq, the differentially expressed genes may play important roles in tissues-specific synthesis, accumulation and modification of flavonoids. CONCLUSION: We present here the de novo transcriptome analysis of E. konishii and the identification of candidate genes involved in biosynthesis and accumulation of flavonoid. In general, these results are an important resource for further research on gene expression, genomic and functional genomics in E. konishii and other related species.

Sequencing of Euscaphis konishii Endocarp Transcriptome Points to Molecular Mechanisms of Endocarp Coloration.

Flower and fruit colors are of vital importance to the ecology and economic market value of plants. The mechanisms of flower and fruit coloration have been well studied, especially among ornamental flower plants and cultivated fruits. As people pay more attention to exocarp coloration, the endocarp coloration in some species has often been ignored. Here, we report on the molecular mechanism of endocarp coloration in three development stages of Euscaphis konishii. The results show that endocarp reddening is closely related to anthocyanin accumulation, and a total of 86,120 unigenes were assembled, with a mean length of 893 bp (N50 length of 1642 bp). We identified a large number of differentially expressed genes associated with endocarp coloration, including anthocyanin biosynthesis, carotenoid biosynthesis, and chlorophyll breakdown. The genes participating in each step of the anthocyanin biosynthesis were found in the transcriptome dataset, but a few genes were found in the carotenoid biosynthesis and chlorophyll breakdown. In addition, the candidate R2R3-MYB transcription factors and candidate glutathione S-transferase transport genes, which likely regulate the anthocyanin biosynthesis, were identified. This study offers a platform for E. konishii functional genomic research and provides a reference for revealing the regulatory mechanisms of endocarp reddening.

Isolation and characterization of a novel native Bacillus thuringiensis strain BRC-HZM2 capable of degrading chlorpyrifos.

Studies were carried out to isolate chlorpyrifos degrading Bacillus thuringiensis (Bt) strains from chlorpyrifos-contaminated samples. Six Bt strains (isolation rate 2.7%) were isolated by modified sodium acetate antibiotic heat treatment, and one novel strain (BRC-HZM2) was selected for further analysis. Phenotype and phylogeny analysis of this strain was conducted on the basis of biochemical reactions, antibiotic sensitivity, 16s rRNA genes, plasmid profile, insecticidal crystal protein profiles, and PCR-RFLP for cry and cyt genes. The degradation rate of chlorpyrifos in liquid culture was estimated during 48 h of incubation for the isolate BRC-HZM2. More than 50% of the initial chlorpyrifos concentration degraded within 12 h, 88.9% after 48 h. These results highlight the potential of the Bt strain for biological control and the bioremediation of environments contaminated with chlorpyrifos.

Use of spent mushroom substrate for production of Bacillus thuringiensis by solid-state fermentation.

The aim of this study was to explore a cost-effective method for the mass production of Bacillus thuringiensis (Bt) by solid-state fermentation. As a locally available agroindustrial byproduct, spent mushroom substrate (SMS) was used as raw material for Bt cultivation, and four combinations of SMS-based media were designed. Fermentation conditions were optimized on the best medium and the optimal conditions were determined as follows: temperature 32 degrees C, initial pH value 6, moisture content 50%, the ratio of sieved material to initial material 1:3, and inoculum volume 0.5 ml. Large scale production of B. thuringiensis subsp. israelensis (Bti) LLP29 was conducted on the optimal medium at optimal conditions. High toxicity (1,487 international toxic units/milligram) and long larvicidal persistence of the product were observed in the study, which illustrated that SMS-based solid-state fermentation medium was efficient and economical for large scale industrial production of Bt-based biopesticides. The cost of production of 1 kg of Bt was approximately US$0.075.

Linking soil phosphorus availability and phosphatase functional genes to coastal marsh erosion: Implications for nutrient cycling and wetland restoration.

Accelerated marsh erosion caused by climate change and human activity may have important implications for nutrient cycling and availability. However, how erosion affects phosphorus (P) transformation and microbial function in subtropical coastal marshes remains largely unknown. Here we assessed soil P fractions, availability and the phoD-harboring bacterial community along a marsh erosion gradient (non-eroded, lightly eroded, and heavily eroded). We showed that marsh erosion caused a shift in P fractions, leading to a decrease in P availability and a reduction in concentrations of labile P, moderately labile P, and stable P by 20 %, 9 %, and 17 % respectively. The abundance and diversity of phoD phosphatase genes decreased dramatically along the erosion gradient and were lower at heavily eroded sites than at non-eroded sites. Marsh erosion reshaped phoD gene community composition, and Corallococcus, Amycolatopsis, and Phaeobacter were identified as the dominant phoD-harboring microbes. Notably, marsh erosion reduced the complexity and stability of the phoD-harboring bacterial network, and heavily eroded sites have fewer network edges and nodes than non-eroded sites. The dynamics of soil P fractions, availability, and phoD-harboring bacterial communities driven by marsh erosion are largely shaped by substrate availability and soil properties (e.g., nutrients, pH, electrical conductivity, and moisture). Additionally, strong linkages between P availability and the abundance and diversity of phosphatase genes following erosion, suggest that phosphatase drives P mineralization and dissolution, and erosion weakens the regulation of P transformation by reshaping the phoD phosphatase gene community. Our findings indicate that marsh erosion alters soil P fractions and phoD-harboring bacterial communities, which weakens microbial regulation of P transformation and availability, thereby significantly reducing soil P pools and availability. Our findings broaden understanding of the impacts of coastal erosion on nutrient balance and ecosystem function, offering valuable perspectives that could inform wetland restoration and coastal management strategies.

Chicken manure application alters microbial community structure and the distribution of antibiotic-resistance genes in rhizosphere soil of Cinnamomum camphora forests.

The distribution of antibiotic-resistance genes (ARGs) in environmental soil is greatly affected by livestock and poultry manure fertilization, the application of manure will lead to antibiotic residues and ARGs pollution, and increase the risk of environmental pollution and human health. Cinnamomum camphora is an economically significant tree species in Fujian Province, China. Here, through high-throughput sequencing analysis, significant differences in the composition of the bacterial community and ARGs were observed between fertilized and unfertilized rhizosphere soil. The application of chicken manure organic fertilizer significantly increased the relative abundance and alpha diversity of the bacterial community and ARGs. The content of organic matter, soluble organic nitrogen, available phosphorus, nitrate reductase, hydroxylamine reductase, urease, acid protease, ß-glucosidase, oxytetracycline, and tetracycline in the soil of C. camphora forests have significant effects on bacterial community and ARGs. Significant correlations between environmental factors, bacterial communities, and ARGs were observed in the rhizosphere soil of C. camphora forests according to Mantel tests. Overall, the findings of this study revealed that chicken manure organic fertilizer application has a significant effect on the bacterial community and ARGs in the rhizosphere soil of C. camphora forests, and several environmental factors that affect the bacterial community and ARGs were identified.

Variation in Community Structure of the Root-Associated Fungi of Cinnamomum camphora Forest.

Plant-associated microbial communities play essential roles in the vegetative cycle, growth, and development of plants. Cinnamomum camphora is an evergreen tree species of the Lauraceae family with high ornamental, medicinal, and economic values. The present study analyzed the composition, diversity, and functions of the fungal communities in the bulk soil, rhizosphere, and root endosphere of C. camphora at different slope positions by high-throughput sequencing. The results showed that the alpha diversity of the fungal communities in the bulk soil and rhizosphere of the downhill plots was relatively higher than those uphill. A further analysis revealed that Mucoromycota, the dominant fungus at the phylum level, was positively correlated with soil bulk density, total soil porosity, mass water content, alkaline-hydrolyzable nitrogen, maximum field capacity, and least field capacity. Meanwhile, the prevalent fungus at the class level, Mortierellomycetes, was positively correlated with total phosphorus and available and total potassium, but negatively with alkaline-hydrolyzable nitrogen. Finally, the assignment of the functional guilds to the fungal operational taxonomic units (OTUs) revealed that the OTUs highly enriched in the downhill samples compared with the uphill samples, which were saprotrophs. Thus, this study is the first to report differences in the fungal community among the different soil/root samples and between C. camphora forests grown at different slope positions. We also identified the factors favoring the root-associated beneficial fungi in these forests, providing theoretical guidance for managing C. camphora forests.

Flavonoid and chromone-rich extract from Euscaphis Konishii Hayata leaf attenuated alcoholic liver injury in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Euscaphis konishii Hayata is a traditional medicinal plant in China, and its leaves are usually used to make dishes for hepatic or gastrointestinal issues by Chinese She nationality. Pharmacological analysis showed that E. konishii leaves contain high levels of flavonoids and chromones with favorable anti-hepatoma effect. AIM OF THE STUDY: The extract from E. konishii leaves was detected to evaluate its chemical composition, and the alcoholic liver injury mice model was adopted to elucidate its hepatoprotective effects. MATERIALS AND METHODS: The total leaf extract from E. konishii was separated by polyamide column to get the flavonoid and chromone-rich extract (FCE). Single compounds from FCE was purified by gel and Sephadex LH-20 chromatography and analyzed by nuclear magnetic resonance (NMR). The chemical component of FCE was confirmed and quantified by HPLC-MS. The OH·, O2-, DPPH and ABTS + free radical assays were adopted to estimate the antioxidant activity of FCE in vitro. The alcohol-fed model mice were established to assess the hepatoprotective capacity of FCE in vivo, through biochemical determination, histopathological analysis, mitochondrial function measurement, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) detection and Western blot determination. RESULTS: 8 flavonoids and 2 chromones were recognized in the FCEextract by both NMR and HPLC-MS. FCE represented strong free radicals scavenging activity in vitro. With oral administration, FCE (50, 100 and 200 mg/kg) dose-dependently decreased the serum levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in alcohol-fed mice. FCE gradually reduced the malondialdehyde (MDA) content, increased the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the alcohol-treated liver tissues. FCE also alleviated the hepatic inflammation, inhibited the hepatocyte apoptosis and lessened the alcohol-induced histological alteration and lipid accumulation in the liver tissues. FCE administration inhibited the overexpression of endoplasmic reticulum (ER) chaperones signaling and unfolded protein response (UPR) pathways to defense the ER-induced apoptosis. Pretreatment with FCE also restored the mitochondrial membrane potentials andadenosine triphosphate (ATP) levels, which in turn suppressed the Cytochrome C release and mitochondria-induced apoptosis. CONCLUSIONS: FCE conferred great protection against alcoholic liver injury, which might be associated with its viability through suppressing reactive oxygen species (ROS) stress and hepatocyte apoptosis.

Field-applied biochar-based MgO and sepiolite composites possess CO2 capture potential and alter organic C mineralization and C-cycling bacterial structure in fertilized soils.

Agricultural soils contribute a significant amount of anthropogenic CO2 emission, a greenhouse gas of global environmental concern. Hence, discovering sustainable materials that can capture CO2 in cultivated soils is paramount. Since the effect of biochar on C mineralization/retention in fertilized soils is unclear, we produced biochar-based MgO and sepiolite-nanocomposites with CO2 capture potential. The field-scale impacts of the modified-biochars were evaluated on net C exchange rate (NCER) periodically for 3 months in fertilized plots. The effects of the modified-biochar on organic-C mineralization, the activities, and dynamics of C-cycling-related 16S rRNA which are unknown, were investigated. Results revealed an initial rapid and higher cumulative CO2 emission from the sole fertilizer treatment (F). Unlike the biochar treatment (BF), the successful incorporation of MgO/Mg(OH)2 nanoparticles into the matrix and surface of biochar, and the potential formation of MgCO3 with soil CO2, mitigated CO2 emission, especially in the MgO-modified biochar (MgOBF), compared to the sepiolite-biochar treatment (SBF). Compared to F and BF, the higher C retention as MgCO3 in the modified biochar treatments led to an increase in cellulase activity, stimulation of key C-cycling-related bacteria, and the expression of genes associated with starch, sucrose, amino sugar, nucleotide sugar, ascorbate, aldarate, cellulose, and chitin degradation, thus, increasing organic C mineralization. Among the modified-biochar treatments, higher C mineralization was recorded in SBF, resulting in increased cumulative CO2 emission, despite its initial capture for up to 42 days. However, MgOBF was effective in capturing soil-derived CO2, despite the increased C mineralization compared to biochar. The changes in soil moisture and temperature significantly regulated NCER. Also, the modified biochars positively influenced the distribution of C-cycling-related bacteria by improving soil pH and available nutrients. Among the modified biochars, the observed higher mitigation effect of MgOBF on NCER indicated that it could be preferably applied in agricultural soils.

Genomes of leafy and leafless Platanthera orchids illuminate the evolution of mycoheterotrophy.

To improve our understanding of the origin and evolution of mycoheterotrophic plants, we here present the chromosome-scale genome assemblies of two sibling orchid species: partially mycoheterotrophic Platanthera zijinensis and holomycoheterotrophic Platanthera guangdongensis. Comparative analysis shows that mycoheterotrophy is associated with increased substitution rates and gene loss, and the deletion of most photoreceptor genes and auxin transporter genes might be linked to the unique phenotypes of fully mycoheterotrophic orchids. Conversely, trehalase genes that catalyse the conversion of trehalose into glucose have expanded in most sequenced orchids, in line with the fact that the germination of orchid non-endosperm seeds needs carbohydrates from fungi during the protocorm stage. We further show that the mature plant of P. guangdongensis, different from photosynthetic orchids, keeps expressing trehalase genes to hijack trehalose from fungi. Therefore, we propose that mycoheterotrophy in mature orchids is a continuation of the protocorm stage by sustaining the expression of trehalase genes. Our results shed light on the molecular mechanism underlying initial, partial and full mycoheterotrophy.

High-Throughput Sequencing Analysis of the Composition and Diversity of the Bacterial Community in Cinnamomum camphora Soil.

Soil bacterial communities and root-associated microbiomes play important roles in the nutrient absorption and healthy growth of host plants. Cinnamomum camphora is an important timber and special economic forest tree species in Fujian Province. In this study, the high-throughput sequencing technique was used to analyze the composition, diversity, and function of the bacterial communities present in the soil from different samples and slope positions of C. camphora. The results of this analysis demonstrated that the related bacterial communities in C. camphora soil were mainly clustered based on sample type. Bacterial alpha diversity in the rhizosphere and bulk soil of C. camphora growing downhill was higher than that of C. camphora growing uphill. At the phylum level, Bacteroidetes, Proteobacteria, Chloroflexi, and Gemmatimonadetes were positively correlated with pH, available phosphorus, total phosphorus, available potassium, and total potassium, while Acidobacteria and Verrucomicrobia were negatively correlated with alkaline-hydrolyzable nitrogen. These results show that there were remarkable differences in the composition, diversity, and function of related bacterial communities between different sample types of C. camphora soil. The slope position had a marked effect on the bacterial communities in the rhizosphere and bulk soil, while the root endosphere remained unaffected.

Genetic diversity and population structure of Euscaphis japonica, a monotypic species.

BACKGROUND: Understanding plant genetic diversity is important for effective conservation and utilization of genetic resources. Euscaphis japonica (Thunb.) Dippel, is a monotypic species with high phenotypic diversity, narrow distribution, and small population size. In this study, we estimated the genetic diversity and population structure of E. japonica using nine natural populations and inter-simple sequence repeat (ISSR) markers. Our results could provide a theoretical reference for future conservation and utilization of E. japonica. RESULTS: We obtained a total of 122 DNA bands, of which 121 (99.18%) were polymorphic. The average number of effective alleles (Ne = 1.4975), Nei's gene diversity index (H = 0.3016), and Shannon's information index (I = 0.4630) revealed that E. japonica possessed a high level of genetic diversity. We observed that E. japonica consisted of both deciduous and evergreen populations. UPGMA tree showed that the evergreen and deciduous E. japonica form a sister group. There is little genetic differentiation among geographic populations based on STRUCTURE analysis. The Dice's similarity coefficient between the deciduous and evergreen populations was low, and the Fst value was high, indicating that these two types of groups have high degree of differentiation. CONCLUSION: Rich genetic diversity has been found in E. japonica, deciduous E. japonica and evergreen E. japonica populations, and genetic variation mainly exists within the population. The low-frequency gene exchange between deciduous and evergreen populations may be the result of the differentiation of deciduous and evergreen populations. We suggest that in-situ protection, seed collection, and vegetative propagation could be the methods for maintenance and conservation of E. japonica populations.

Genome-Wide Identification and Co-Expression Analysis of ARF and IAA Family Genes in Euscaphis konishii: Potential Regulators of Triterpenoids and Anthocyanin Biosynthesis.

Euscaphis konishii is an evergreen plant that is widely planted as an industrial crop in Southern China. It produces red fruits with abundant secondary metabolites, giving E. konishii high medicinal and ornamental value. Auxin signaling mediated by members of the AUXIN RESPONSE FACTOR (ARF) and auxin/indole-3-acetic acid (Aux/IAA) protein families plays important roles during plant growth and development. Aux/IAA and ARF genes have been described in many plants but have not yet been described in E. konishii. In this study, we identified 34 EkIAA and 29 EkARF proteins encoded by the E. konishii genome through database searching using HMMER. We also performed a bioinformatic characterization of EkIAA and EkARF genes, including their phylogenetic relationships, gene structures, chromosomal distribution, and cis-element analysis, as well as conserved motifs in the proteins. Our results suggest that EkIAA and EkARF genes have been relatively conserved over evolutionary history. Furthermore, we conducted expression and co-expression analyses of EkIAA and EkARF genes in leaves, branches, and fruits, which identified a subset of seven EkARF genes as potential regulators of triterpenoids and anthocyanin biosynthesis. RT-qPCR, yeast one-hybrid, and transient expression analyses showed that EkARF5.1 can directly interact with auxin response elements and regulate downstream gene expression. Our results may pave the way to elucidating the function of EkIAA and EkARF gene families in E. konishii, laying a foundation for further research on high-yielding industrial products and E. konishii breeding.