Genome-wide characterization of post-transcriptional processes related to wood formation in Dalbergia odorifera.

BACKGROUND: Alternative polyadenylation (APA), alternative splicing (AS), and long non-coding RNAs (lncRNAs) play regulatory roles in post-transcriptional processes in plants. However, little is known about their involvement in xylem development in Dalbergia odorifera, a valuable rosewood species with medicinal and commercial significance. We addressed this by conducting Isoform Sequencing (Iso-Seq) using PacBio's SMRT technology and combined it with RNA-seq analysis (RNA sequencing on Illumina platform) after collecting xylem samples from the transition zone and the sapwood of D. odorifera. RESULTS: We identified 14,938 full-length transcripts, including 9,830 novel isoforms, which has updated the D. odorifera genome annotation. Our analysis has revealed that 4,164 genes undergo APA, whereas 3,084 genes encounter AS. We have also annotated 118 lncRNAs. Furthermore, RNA-seq analysis identified 170 differential alternative splicing (DAS) events, 344 genes with differential APA site usage (DE-APA), and 6 differentially expressed lncRNAs in the transition zone when compared to the sapwood. AS, APA, and lncRNAs are differentially regulated during xylem development. Differentially expressed APA genes were enriched for terpenoid and flavonoid metabolism, indicating their role in the heartwood formation. Additionally, DE-APA genes were associated with cell wall biosynthesis and terpenoid metabolism, implying an APA's role in wood formation. A DAS gene (involved in chalcone accumulation) with a significantly greater inclusion of the last exon in the transition zone than in the sapwood was identified. We also found that differentially expressed lncRNAs targeted the genes related to terpene synthesis. CONCLUSIONS: This study enhances our understanding of the molecular regulatory mechanisms underlying wood formation in D. odorifera, and provides valuable genetic resources and insights for its molecular-assisted breeding.

Physio-biochemical and metabolomic responses of the woody plant Dalbergia odorifera to salinity and waterlogging.

BACKGROUND: Trees have developed a broad spectrum of molecular mechanisms to counteract oxidative stress. Secondary metabolites via phenolic compounds emblematized the hidden bridge among plant kingdom, human health, and oxidative stress. Although studies have demonstrated that abiotic stresses can increase the production of medicinal compounds in plants, research comparing the efficiency of these stresses still needs to be explored. Thus, the present research paper provided an exhaustive comparative metabolomic study in Dalbergia odorifera under salinity (ST) and waterlogging (WL). RESULTS: High ST reduced D. odorifera's fresh biomass compared to WL. While WL only slightly affected leaf and vein size, ST had a significant negative impact. ST also caused more significant damage to water status and leaflet anatomy than WL. As a result, WL-treated seedlings exhibited better photosynthesis and an up-regulation of nonenzymatic pathways involved in scavenging reactive oxygen species. The metabolomic and physiological responses of D. odorifera under WL and salinity ST stress revealed an accumulation of secondary metabolites by the less aggressive stress (WL) to counterbalance the oxidative stress. Under WL, more metabolites were more regulated compared to ST. ST significantly altered the metabolite profile in D. odorifera leaflets, indicating its sensitivity to salinity. WL synthesized more metabolites involved in phenylpropanoid, flavone, flavonol, flavonoid, and isoflavonoid pathways than ST. Moreover, the down-regulation of L-phenylalanine correlated with increased p-coumarate, caffeate, and ferulate associated with better cell homeostasis and leaf anatomical indexes under WL. CONCLUSIONS: From a pharmacological and medicinal perspective, WL improved larger phenolics with therapeutic values compared to ST. Therefore, the data showed evidence of the crucial role of medical tree species' adaptability on ROS detoxification under environmental stresses that led to a significant accumulation of secondary metabolites with therapeutic value.

Changes in the physiological activity of parenchyma cells in Dalbergia odorifera xylem and its relationship with heartwood formation.

BACKGROUND: The formation of a tree's heartwood gives the wood properties such as natural decay resistance and aesthetic color, and often directly determines the value of wood products. Regulating the quantity and quality of heartwood is of great importance to the use of wood. However, the mechanism of heartwood formation has been poorly understood. RESULTS: Using Dalbergia odorifera as the study species, the number of starch grains, the morphology of the nuclei, the changes in the content of water and secondary metabolites were observed continuously in the radial direction of the xylem. The results show that from the outer toward inner sapwood, the starch grains are abundant, the length to diameter ratio of the nuclei is decreasing, and the morphology changes from elongated elliptical and then to round. In the outer transition zone, the starch grains begin to decrease abruptly and the nuclei shrink at a slower rate, with a radial width of approximately 2 mm. In the inner transition zone, the heartwood color begins to appear, the starch grains disappear and a few nuclei with reduced fluorescence are present, with a radial width of approximately 1 mm. Heartwood formation after complete disappearance of the nuclei. The moisture content of the heartwood is higher than that of the sapwood, and the inner transition zone is where the content rises. The secondary metabolites of the heartwood begin to accumulate in large quantities in the inner transition zone. CONCLUSION: Based on the physiological changes of parenchyma cells in the xylem, the radial width of the transition zone of Dalbergia odorifera is clearly defined as approximately 3 mm. Both the water and secondary metabolite abrupt changes occur at the final stage of programmed cell death, and neither is a direct cause of programmed cell death in parenchyma cells.

Identification of the effective α-amylase inhibitors from Dalbergia odorifera: Virtual screening, spectroscopy, molecular docking, and molecular dynamic simulation.

Inhibiting the activity of α-amylase has been considered as one efficient way to prevent and treat type 2 diabetes recently. Dalbergia odorifera, a kind of Leguminosae plant, has a positive therapeutic effect on type 2 diabetes, possibly contributing by some constituents that can inhibit the activity of α-amylase. In this study, we found that eriodictyol was one potential constituent through virtual screening. The interaction mode between eriodictyol and α-amylase was elucidated by molecular docking, multi-spectroscopic analysis, and molecular dynamic simulation. The results revealed that eriodictyol quenched the intrinsic fluorescence of α-amylase, and the quenching mode was static quenching. Eriodictyol could spontaneously interact with α-amylase, mostly stabilized and influenced by the hydrophobic interaction, while the binding sites (n) was 1.13 ± 0.07 and binding constant (Kb) was (1.43 ± 0.14) × 105 at 310 K, respectively. In addition, FT-IR and CD had been applied to identify that eriodictyol can trigger the conformational change of α-amylase. Taken together, the results provided some experimental data for developing new α-amylase inhibitors from Dalbergia odorifera, which may further prevent and treat diabetes and diabetes complications.

The physiological and molecular mechanisms of N transfer in Eucalyptus and Dalbergia odorifera intercropping systems using root proteomics.

BACKGROUND: The mixing of Eucalyptus with N2-fixing trees species (NFTs) is a frequently successful and sustainable cropping practice. In this study, we evaluated nitrogen (N) transfer and conducted a proteomic analysis of the seedlings of Eucalyptus urophylla × E. grandis (Eucalyptus) and an NFT, Dalbergia (D.) odorifera, from intercropping and monocropping systems to elucidate the physiological effects and molecular mechanisms of N transfer in mixed Eucalyptus and D. odorifera systems. RESULTS: N transfer occurred from D. odorifera to Eucalyptus at a rate of 14.61% in the intercropping system, which increased N uptake and growth in Eucalyptus but inhibited growth in D. odorifera. There were 285 and 288 differentially expressed proteins by greater than 1.5-fold in Eucalyptus and D. odorifera roots with intercropping vs monoculture, respectively. Introduction of D. odorifera increased the stress resistance ability of Eucalyptus, while D. odorifera stress resistance was increased by increasing levels of jasmonic acid (JA). Additionally, the differentially expressed proteins of N metabolism, such as glutamine synthetase nodule isozyme (GS), were upregulated to enhance N competition in Eucalyptus. Importantly, more proteins were involved in synthetic pathways than in metabolic pathways in Eucalyptus because of the benefit of N transfer, and the two groups of N compound transporters were found in Eucalyptus; however, more functional proteins were involved in metabolic degradation in D. odorifera; specifically, the molecular mechanism of the transfer of N from D. odorifera to Eucalyptus was explained by proteomics. CONCLUSIONS: Our study suggests that N transfer occurred from D. odorifera to Eucalyptus and was affected by the variations in the differentially expressed proteins. We anticipate that these results can be verified in field experiments for the sustainable development of Eucalyptus plantations.

Nutrient uptake and assimilation in fragrant rosewood (Dalbergia odorifera T.C. Chen) seedlings in growing media with un-composted spent mushroom residue.

The purpose of this study was to detect nutrient uptake and assimilation in woody plants subjected to growing media with some peat replaced by spent mushroom residue (SMR). Fragrant rosewood (Dalbergia odorifera T.C. Chen) seedlings were cultured in five types of growing media with SMR and peat in volumetric proportions of 0% (control), 25%, 50%, 75%, and 100%. With the increase of SMR proportion, ammonium- and nitrate-nitrogen (N) concentrations declined but available phosphorus (P) concentration and electrical conductance both increased. Seedlings in the full SMR substrates showed obvious mortality. Seedlings in substrates with SMR in proportions higher than 25% showed symptoms of excessive N and P toxicities. The utilization efficiency for P was highest in the 25% SMR growing-media. Mineral N in substrates had a positive relationship with growth and biomass but not with glutamine synthetase activity. Available P was negatively related with acid phosphatase activity in both leaves and roots. Un-composted SMR can replace 25% of peat in growing media for fragrant rosewood seedlings, benefitting P uptake and assimilation.

Serine protease inhibitors and activators from Dalbergia tonkinensis species.

The vulnerable plant Dalbergia tonkinensis Prain is a rare species in Vietnam. In the course of our studies on biologically active plants, we performed serine protease enzyme screenings. The results suggest that at concentrations of 25-250 ng/mL, methanol extracts of leaf and root, root ethanol extract and its dichloromethane fraction, and heartwood water decoction extract can serve as useful sources to stimulate trypsin enzyme activity. In addition, water decoction extracts of leaf and stem bark may explain unknown ethno-pharmacology due to the high inhibitory effects in enzyme assays using trypsin, chymotrypsin, and elastase. Among 23 isolated compounds and two semi-synthetic derivatives tested, quercetin (17) inhibits the activities of trypsin and chymotrypsin with IC50 9.7 µM. Flavonoids categorized as flavanone, isoflavanone, flavone, isoflavone, pretocarpan, aurone, and neoflavanone demonstrated variable activities. Several substitutions are closely correlated with protease actions, including hydroxylation at C-3 and C-3' in flavone and C-5 and C-3' in isoflavone, hydroxylation at C-3, C-5 and C-3', carboxylation at C-6 and C-8, and 7-substitution in flavanone; 7-substitution and methoxylation at C-3' in isoflavanone; and lactone ring opening in neoflavanone. In the assessment of casein cleavage, at a dose of 25 ng/mL, leaf water decoction extract demonstrates an inhibitory effect on casein cleavage by trypsin, whereas ethanol and methanol extracts of the root caused activation.

Selection and Validation of Reference Genes for Gene Expression Studies by RT-PCR in Dalbergia odorifera.

Perennial tree Dalbergia odorifera T. Chen could form the precious heartwood used to produce chinese traditional medicine, rosewood furniture and fragrances. However the formation of heartwood is time-consuming and low efficient, leading to the severe destruction of its wild resources. Thus, it is urgent to study the molecular mechanism of heartwood formation in D. odorifera. But till now, there is no report about the reference gene selection in this species. In this study, the expression stability of nine candidate reference genes were evaluated across different tissues and stems treated by wound and chemical stimulators. Four algorithms were applied to obtain the robust genes. The results support HIS2, GAPDH, and CYP to be the most stable reference genes in samples under different wound treatments while DNAj was the least stable. In different tissues, HIS2, UBQ, and RPL were the most stable reference genes while DNAj was the least stable. The selected reference genes were validated through the normalization of the qRT-PCR data of six heartwood related genes in terpene biosynthesis pathway and ethylene signal pathway. The results showed that their expression levels were accurate when they were normalized by the most stable reference gene HIS2, or by the combination of the two or three most stable reference genes. These results demonstrated that these selected reference genes are reliable.

Characterization of the complete chloroplast genome sequence of Dalbergia species and its phylogenetic implications.

The pantropical plant genus Dalbergia comprises approximately 250 species, most of which have a high economic and ecological value. However, these species are among the most threatened due to illegal logging and the timber trade. To enforce protective legislation and ensure effective conservation of Dalbergia species, the identity of wood being traded must be accurately validated. For the rapid and accurate identification of Dalbergia species and assessment of phylogenetic relationships, it would be highly desirable to develop more effective DNA barcodes for these species. In this study, we sequenced and compared the chloroplast genomes of nine species of Dalbergia. We found that these chloroplast genomes were conserved with respect to genome size, structure, and gene content and showed low sequence divergence. We identified eight mutation hotspots, namely, six intergenic spacer regions (trnL-trnT, atpA-trnG, rps16-accD, petG-psaJ, ndhF-trnL, and ndhG-ndhI) and two coding regions (ycf1a and ycf1b), as candidate DNA barcodes for Dalbergia. Phylogenetic analyses based on whole chloroplast genome data provided the best resolution of Dalbergia, and phylogenetic analysis of the Fabaceae showed that Dalbergia was sister to Arachis. Based on comparison of chloroplast genomes, we identified a set of highly variable markers that can be developed as specific DNA barcodes.

Lead Tolerance and its Accumulation by a Tree Legume: Dalbergia sissoo DC.

Dalbergia sissoo DC, a leguminous tropical timber tree has been investigated against the Pb toxicity; under the Pb-stress, plant's morphology, biochemical parameters and genomic template stability (GTS) screened in vitro. At the optimum Pb tolerance level (150 mg L-1), plant's defense mechanism-superoxide dismutase, catalase, ascorbate peroxidases and proline could trigger to achieve optimum vegetative growth with minimum fluctuations of the GTS. Further, D. sissoo roots could accumulate 2399.8 ± 16 mg kg-1 Pb. Scanning electron microscopy and energy dispersive X-ray spectrometer analysis also revealed the deposition of Pb in root tissues. In a 1 year pot experiment with Pb-contaminated soil, the plants exhibited normal growth, and Pb accumulation significantly enhanced by the amalgamation of citric acid in the soil. Thus, the tree may prove as a potential candidate for Pb phytostabilization.

Two-way transfer of nitrogen between Dalbergia odorifera and its hemiparasite Santalum album is enhanced when the host is effectively nodulated and fixing nitrogen.

Nutrient translocation from a host plant is vital to the growth and survival of its root parasitic plant, but few studies have investigated whether a parasitic plant is also able to transfer nutrients to its host. The role of N2-fixation in nitrogen (N) transfer between 7-month-old Dalbergia odorifera T. Chen nodulated with Bradyrhizobium elkanii DG and its hemiparasite Santalum album Linn. was examined by external (15)N labeling in a pot study. Four paired treatments were used, with (15)N given to either host or hemiparasite and the host either nodulated or grown on combined N. N2-fixation supplied 41-44% of total N in D. odorifera. Biomass, N and (15)N contents were significantly greater in both nodulated D. odorifera and S. album grown with paired nodulated D. odorifera. Significantly higher total plant (15)N recovery was in N donor D. odorifera (68-72%) than in N donor S. album (42-44%), regardless of the nodulation status in D. odorifera. Nitrogen transfer to S. album was significantly greater (27.8-67.8 mg plant(-1)) than to D. odorifera (2.0-8.9 mg plant(-1)) and 2.4-4.5 times greater in the nodulated pair than in the non-nodulated pair. Irrespective of the nodulation status, S. album was always the N-sink plant. The amount of two-way N transfer was increased by the presence of effective nodules, resulting in a greater net N transfer (22.6 mg plant(-1)) from host D. odorifera to hemiparasite S. album. Our results may provide N management strategies for D. odorifera/S. album mixed plantations in the field.

A systems biology approach to understanding the mechanisms of action of chinese herbs for treatment of cardiovascular disease.

Traditional Chinese Medicine (TCM) involves a broad range of empirical testing and refinement and plays an important role in the health maintenance for people all over the world. However, due to the complexity of Chinese herbs, a full understanding of TCM's action mechanisms is still unavailable despite plenty of successful applications of TCM in the treatment of various diseases, including especially cardiovascular diseases (CVD), one of the leading causes of death. Thus in the present work, by incorporating the chemical predictors, target predictors and network construction approaches, an integrated system of TCM has been constructed to systematically uncover the underlying action mechanisms of TCM. From three representative Chinese herbs, i.e., Ligusticum chuanxiong Hort., Dalbergia odorifera T. Chen and Corydalis yanhusuo WT Wang which have been widely used in CVD treatment, by combinational use of drug absorption, distribution, metabolism and excretion (ADME) screening and network pharmacology techniques, we have generated 64 bioactive ingredients and identified 54 protein targets closely associated with CVD, of which 29 are common targets (52.7%) of the three herbs. The result provides new information on the efficiency of the Chinese herbs for the treatment of CVD and also explains one of the basic theories of TCM, i.e., "multiple herbal drugs can treat one disease". The predicted potential targets were then mapped to target-disease and target-signal pathway connections, which revealed the relationships of the active ingredients with their potential targets, diseases and signal systems. This means that for the first time, the action mechanism of these three important Chinese herbs for the treatment of CVD is uncovered, by generating and identifying both their active ingredients and novel targets specifically related to CVD, which clarifies some of the common conceptions in TCM, and thus provides clues to modernize such specific herbal medicines.

Phenotypic plasticity to light of two congeneric trees from contrasting habitats: Brazilian Atlantic Forest versus cerrado (savanna).

The Brazilian Atlantic Forest is a typically multi-layer tropical forest, while cerrado (savanna) is a patchy habitat with different physiognomy. Despite these differences, both habitats have high light heterogeneity. Functional traits of Dalbergia nigra and D. miscolobium from the Atlantic Forest and cerrado, respectively, were evaluated under shade (25% of full sunlight) and full sunlight in a nursery experiment. We hypothesised that both species should benefit from high phenotypic plasticity in relation to light. Plasticity was estimated using the relative distance phenotypic index (RDPI). D. miscolobium had lower shoot growth under both light conditions, suggesting it has low competitive capacity in the forest environment, which could explain its limited ability to expand over areas of Atlantic Forest. The studied species exhibited photoprotection strategies under high light and improved light capture under low light. Stomatal conductance, ETR(max) (maximum electron transport rate), PPFD(sat) (saturating photosynthetically active photon flux density), chlorophyll and carotenoid content had higher RDPI than stem morphological traits. Although both species showed considerable phenotypic plasticity, D. miscolobium had higher RDPI for eight of 11 evaluated traits. This high plasticity could be one of the factors that explain the occurrence of this species in a wide range of environmental conditions, from open grassland to dense woodlands, and it could also reflect its adaptation to high light. D. nigra also had considerable plasticity and good growth performance in both shade and full sunlight, but its absence in areas of cerrado suggests that factors other than light limit its occurrence in these habitats.

Aluminium tolerance and high phosphorus efficiency helps Stylosanthes better adapt to low-P acid soils.

BACKGROUND AND AIMS: Stylosanthes spp. (stylo) is one of the most important pasture legumes used in a wide range of agricultural systems on acid soils, where aluminium (Al) toxicity and phosphorus (P) deficiency are two major limiting factors for plant growth. However, physiological mechanisms of stylo adaptation to acid soils are not understood. METHODS: Twelve stylo genotypes were surveyed under field conditions, followed by sand and nutrient solution culture experiments to investigate possible physiological mechanisms of stylo adaptation to low-P acid soils. KEY RESULTS: Stylo genotypes varied substantially in growth and P uptake in low P conditions in the field. Three genotypes contrasting in P efficiency were selected for experiments in nutrient solution and sand culture to examine their Al tolerance and ability to utilize different P sources, including Ca-P, K-P, Al-P, Fe-P and phytate-P. Among the three tested genotypes, the P-efficient genotype 'TPRC2001-1' had higher Al tolerance than the P-inefficient genotype 'Fine-stem' as indicated by relative tap root length and haematoxylin staining. The three genotypes differed in their ability to utilize different P sources. The P-efficient genotype, 'TPRC2001-1', had superior ability to utilize phytate-P. CONCLUSIONS: The findings suggest that possible physiological mechanisms of stylo adaptation to low-P acid soils might involve superior ability of plant roots to tolerate Al toxicity and to utilize organic P and Al-P.

Simultaneous determination of 10 major flavonoids in Dalbergia odorifera by high performance liquid chromatography.

A reversed-phase liquid chromatographic method was developed for the simultaneous quantification of 10 major flavonoids, namely butin, (3R)-4'-methoxy-2',3,7-trihydroxyisoflavanone, liquiritigenin, melanettin, violanone, vistitone, formononetin, dalbergin, sativanone and medicarpin in the heartwood of Dalbergia odorifera, an important traditional Chinese medicine. Samples were extracted with 60% methanol. The optimal conditions of separation and detection were achieved on an Agilent Zorbax SB-C18 column (250 mm x 4.6 mm, 5 microm) with a gradient of acetonitrile and 0.3% (v/v) aqueous acetic acid, at a flow rate of 0.8 ml/min, detected at 275 nm. The complete separation was obtained within 55 min for the 10 target compounds. All calibration curves showed good linearity (r2>0.999) within test ranges. The assay was reproducible with overall intra- and inter-day variation of less than 3%. The mean recovery of the method was 100+/-10%, with R.S.D. less than 5%. The current assay method was considered to be suitable for the quality control of D. odorifera samples and could be readily utilized for the determination of the active principles present in this medicinal herb.

Identification of RAPD markers, in situ DNA content and structural chromosomal diversity in some legumes of the mangrove flora of Orissa.

Randomly amplified polymorphic DNA (RAPD) markers, karyotypes and 4C DNA content were analyzed in five legume mangroves belonging to the sub-family Papilinoideae (Dalbergia spinosa, Derris heterophylla and D. indica) and Caesalpinioideae (Caesalpinia crista, Cynometra ramiflora) of the family Fabaceae to establish the genetic variability and phylogenetic affinities. Somatic chromosome numbers were reported for the first time in D. spinosa (2n = 20), C. ramiflora (2n = 26) and D. heterophylla (2n = 24) with reconfirmation of the somatic chromosome number in D. indica (2n = 22) and C. crista (2n = 24). Significant intergeneric and interspecific variation of 4C DNA content was observed and that varied from 8.970 pg in C. ramiflora to 28.730 pg in D. indica. From the RAPD analysis, the dendogram showed clustering of Caesalpinia crista and Cynomitra ramiflora into one group (81.80). In the second groups Derris indica and Derris heterophylla were more similar (83.10) than Dalbergia spinosa (85.80). Species-specific DNA markers (900 bp) obtained in D. spinosa from OPN15; 700 and 2000 bp in C. ramiflora from OPN4 and 400 and 800 bp in D. heterophylla and 500 bp DNA fragment in C. crista obtained from OPN-11 were found characteristic RAPD markers of these species. C. crista found more closer affinity to C. ramiflora of the sub-family Caesalpinioideae [genetic distance (1-F) = 0.847]. Derris indica showed closer genetic relation with D. heterophylla [genetic distance (1-F) = 0.856] than D. spinosa [genetic distance (1-F) = 0.876] where Derris and Dalbergia belongs to the sub-family Papilionoideae. By employing these markers the present study has helped to resolve the relationship between the taxonomically diverse leguminous mangroves and study their ability to coexist with mangroves that would shed light on the evolution of mangroves from terrestrial species.