Reference Genes Selection and Validation for Cinnamomum burmanni by Real-Time Quantitative Polymerase Chain Reaction.

In recent years, the field of biology has witnessed a surge of interest in genomics research due to the advancements in biotechnology. Gene expression pattern analysis plays a crucial role in this research, as it enables us to understand the regulatory mechanism of gene expression and the associated biological processes. Real-time quantitative polymerase chain reaction (q-PCR) is an efficient method to analyze the gene expression patterns, for which accuracy relies on the standardized analysis of reference genes. However, numerous studies have shown that no reference gene is universal in all conditions, so screening a suitable reference gene under certain conditions is of great importance. Cinnamomum burmannii (C. burmannii) is rich in volatile components and has high medicinal and economic value. However, knowledge of the screening of reference genes for the gene expression analysis of C. burmannii is insufficient. Aiming at this problem, we evaluated and screened the reference genes in C. burmannii under different experimental conditions, including different abiotic stresses (Cold-treated, PEG-treated and Nacl-treated), different tissues, leaves at different developmental stages and different chemical types. In this study, different algorithms (∆Ct, geNorm, NormFinder and BestKeeper) were used to evaluate the stability of the candidate reference genes, and RefFinder further merged the output data to screen out the optimum reference gene under various experimental conditions in C. burmannii. The results showed that the optimal reference gene number for gene standardization was 2 under different experimental conditions. RPL27|RPS15 was the most suitable combination under the Nacl-treated and PEG-treated samples. RPL27|APT was the optimum combination under the Cold-treated samples. The optimal combinations of other samples were EF1α|ACT7 for different tissues, eIF-5A|Gllα for different borneol clones in C. burmannii, RPS15|ACT7 for leaves at different developmental stages and RPS15|TATA for all samples. Additionally, two terpenoid synthesis-related genes (CbWRKY4 and CbDXS2) were standardized to verify the feasibility of the selected reference genes under different experimental conditions. This study will be helpful for the subsequent molecular genetic mechanism study of C. burmannii.

Revelation of enzyme/transporter-mediated metabolic regulatory model for high-quality terpene accumulation in developing fruits of Lindera glauca.

Lindera glauca with rich resource and fruit terpene has emerged as potential material for utilization in China, but different germplasms show a variation for essential oil content and volatile profiling. This work aimed to determine key regulators (enzymes or transporters) and unravel mechanism of governing high production of essential oil of L. glauca fruit (EO-LGF). Temporal analysis of fruit growth and EO-LGF accumulation (yield, volatile compounds and contents) during development revealed a notable change in the contents of EO-LGF and its 45 compounds in developing fruits, and the major groups were monoterpene and sesquiterpene, showing good antioxidant and antimicrobial activities. To highlight molecular mechanism that govern such difference in terpene content and compound in developing fruits, Genome-wide assay was used to annotate 104 genes for terpene-synthesis pathway based on recent transcriptome data, and the comparative associations of terpene accumulative amount with gene transcriptional level were conducted on developing fruits to identify some crucial determinants (enzymes and transporters) with metabolic regulation model for high-quality terpene accumulation, involving in carbon allocation (sucrose cleavage, glycolysis and OPP pathway), metabolite transport, isoprene precursor production, C5-unit formation (MEP and MVA pathways), and mono-/sesqui-terpene synthesis. Our findings may present strategy for engineering terpene accumulation for utilization.

Oxidative Stress-Mediated Repression of Virulence Gene Transcription and Biofilm Formation as Antibacterial Action of Cinnamomum burmannii Essential Oil on Staphylococcus aureus.

This work aimed to identify the chemical compounds of Cinnamomum burmannii leaf essential oil (CBLEO) and to unravel the antibacterial mechanism of CBLEO at the molecular level for developing antimicrobials. CBLEO had 37 volatile compounds with abundant borneol (28.40%) and showed good potential to control foodborne pathogens, of which Staphylococcus aureus had the greatest inhibition zone diameter (28.72 mm) with the lowest values of minimum inhibitory concentration (1.0 µg/mL) and bactericidal concentration (2.0 µg/mL). To unravel the antibacterial action of CBLEO on S. aureus, a dynamic exploration of antibacterial growth, material leakage, ROS formation, protein oxidation, cell morphology, and interaction with genome DNA was conducted on S. aureus exposed to CBLEO at different doses (1/2-2×MIC) and times (0-24 h), indicating that CBLEO acts as an inducer for ROS production and the oxidative stress of S. aureus. To highlight the antibacterial action of CBLEO on S. aureus at the molecular level, we performed a comparative association of ROS accumulation with some key virulence-related gene (sigB/agrA/sarA/icaA/cidA/rsbU) transcription, protease production, and biofilm formation in S. aureus subjected to CBLEO at different levels and times, revealing that CBLEO-induced oxidative stress caused transcript suppression of virulence regulators (RsbU and SigB) and its targeted genes, causing a protease level increase destined for the biofilm formation and growth inhibition of S. aureus, which may be a key bactericidal action. Our findings provide valuable information for studying the antibacterial mechanism of essential oil against pathogens.

Native promoter-mediated transcriptional regulation of crucial oleosin protein OLE1 from Prunus sibirica for seed development and high oil accumulation.

Oleosin (OLE) is vital to stabilize lipid droplet for seed triacylglycerol (TAG) storage. This work aimed to determine key OLE and to unravel mechanism that governed seed oil accumulation of Prunus sibirica for developing biodiesel. An integrated assay of global identification of LD-related protein and the cross-accessions/developing stages comparisons associated with oil accumulative amount and OLE transcript level was performed on seeds of 12 plus trees of P. sibirica to identify OLE1 (15.5 kDa) as key oleosin protein crucial for high seed oil accumulation. The OLE1 gene and its promoter were cloned from P. sibirica seeds, and overexpression of PsOLE1 in Arabidopsis was conducted under the controls of native promoter and constitutive CaMV35S promoter, respectively. PsOLE1 promoter had seed-specific cis-elements and showed seed specificity, by which PsOLE1 was specifically expressed in seeds. Ectopic overexpression of PsOLE1, especially driven by its promoter, could facilitate seed development and oil accumulation with an increase in unsaturated FAs, and upregulate transcript of TAG assembly enzymes, but suppress transcript of LD/TAG-hydrolyzed lipases and transporters, revealing a role of native promoter-mediated transcription of PsOLE1 in seed development and oil accumulation. PsOLE1 and its promoter have considerable potential for engineering oil accumulation in oilseed plants.

Efficacy and safety of acupoint application in the treatment of ulcerative colitis: A systematic review and meta-analysis.

BACKGROUND: The efficacy of acupoint application in the treatment of ulcerative colitis (UC) is still controversial. The purpose of this study is to systematically evaluate the clinical efficacy and safety of acupoint application in the treatment of ulcerative colitis. METHODS: The databases of China National Knowledge Infrastructure (CNKI), Chinese Biology Medicine (CBM), VIP, Wanfang, Embase, PubMed, the Cochrane Library and Web of Science were searched. The time limit was from the establishment of the database to July 2022. The published randomized controlled trials of acupoint application in the treatment of UC were analyzed by meta-analysis and trial sequential analysis. RESULTS: A total of 13 studies were included, with a total sample size of 878 cases. Compared with conventional western medicine, acupoint application can effectively improve the effective rates of clinical comprehensive (risk ratio [RR] 1.13, 95% confidence interval [CI] 1.06-1.20, P = .0003), syndrome (RR 1.13, 95% CI 1.03-1.24, P = .009), and interleukin-4 (IL-4) (mean differences 2.62, 95% CI 1.96-3.28, P < .00001) in the treatment of UC, and reduce interferon-γ (mean differences -5.38, 95% CI -6.81 to -3.94, P < .00001). The effective rates of colonoscopy (RR 0.94, 95% CI 0.84-1.05, P = .25), pathological examination (RR 1.04, 95% CI 0.90-1.20, P = .60) and rate of adverse reaction (RR 0.55, 95% CI 0.25-1.21, P = .14) were the same. Trial sequential analysis indicated that the benefits of effective rates of clinical comprehensive and syndrome, IL-4, and interferon-γ were conclusive. Harbord regression showed no publication bias (P = .98). The evaluation of evidence quality suggested that the evidence quality of effective rates of clinical comprehensive and syndrome was moderate and the evidence quality of other indicators was low or very low. CONCLUSION: Acupoint application is a safe and effective method for the treatment of UC, and has the prospect of clinical application.

Determination of superior Pistacia chinensis accession with high-quality seed oil and biodiesel production and revelation of LEC1/WRI1-mediated high oil accumulative mechanism for better developing woody biodiesel.

BACKGROUND: Based on our previous studied on different provenances of Pistacia chinensis, some accessions with high quality and quantity of seed oils has emerged as novel source of biodiesel. To better develop P. chinensis seed oils as woody biodiesel, a concurrent exploration of oil content, FA profile, biodiesel yield, and fuel properties was conducted on the seeds from 5 plus germplasms to determine superior genotype for ideal biodiesel production. Another vital challenge is to unravel mechanism that govern the differences in oil content and FA profile of P. chinensis seeds across different accessions. FA biosynthesis and oil accumulation of oil plants are known to be highly controlled by the transcription factors. An integrated analysis of our recent transcriptome data, qRT-PCR detection and functional identification was performed as an attempt to highlight LEC1/WRI1-mediated transcription regulatory mechanism for high-quality oil accumulation in P. chinensis seeds. RESULTS: To select ideal germplasm and unravel high oil accumulative mechanism for developing P. chinensis seed oils as biodiesel, five plus trees (accession PC-BJ/PC-AH/PC-SX/PC-HN/PC-HB) with high-yield seeds were selected to assess the variabilities in weight, oil content, FA profile, biodiesel yield and fuel property, revealing a variation in the levels of seed oil (50.76-60.88%), monounsaturated FA (42.80-70.72%) and polyunsaturated FA (18.78-43.35%), and biodiesel yield (84.98-98.15%) across different accessions. PC-HN had a maximum values of seed weight (26.23 mg), oil (60.88%) and biodiesel yield (98.15%), and ideal proportions of C18:1 (69.94%), C18:2 (17.65%) and C18:3 (1.13%), implying that seed oils of accession PC-HN was the most suitable for ideal biodiesel production. To highlight molecular mechanism that govern such differences in oil content and FA profile of different accessions, a combination of our recent transcriptome data, qRT-PCR detection and protein interaction analysis was performed to identify a pivotal role of LEC1/WRI1-mediated transcription regulatory network in high oil accumulation of P. chinensis seeds from different accessions. Notably, overexpression of PcWRI1 or PcLEC1 from P. chinensis seeds in Arabidopsis could facilitate seed development and upregulate several genes relevant for carbon flux allocation (plastidic glycolysis and acetyl-CoA generation), FA synthesis, TAG assembly and oil storage, causing an increase in seed oil content and monounsaturated FA level, destined for biodiesel fuel property improvement. Our findings may present strategies for better developing P. chinensis seed oils as biodiesel feedstock and bioengineering its high oil accumulation. CONCLUSIONS: This is the first report on the cross-accessions assessments of P. chinensis seed oils to determine ideal accession for high-quality biodiesel production, and an effective combination of PcWRI1 or PcLEC1 overexpression, morphological assay, oil accumulation and qRT-PCR detection was applied to unravel a role of LEC1/WRI1-mediated regulatory network for oil accumulation in P. chinensis seeds, and to highlight the potential application of PcWRI1 or PcLEC1 for increasing oil production. Our finding may provide new strategies for developing biodiesel resource and molecular breeding.

Evaluation of oil accumulation and biodiesel property of Lindera glauca fruits among different germplasms and revelation of high oil producing mechanism for developing biodiesel.

BACKGROUND: Lindera glauca with rich resource and fruit oil has emerged as novel source of biodiesel in China, but different germplasms show a variation for fruit oil content and FA profile. To develop L. glauca fruit oils as biodiesel, a concurrent exploration of oil content, FA composition, biodiesel yield, fuel property and prediction model construction was conducted on the fruits from 8 plus germplasms to select superior genotype for ideal biodiesel production. Another vital focus was to highlight mechanism that govern the differences in oil content and FA profile of different germplasms. The cross-accessions comparisons associated with oil-synthesized gene transcriptional level and oil accumulative amount led to the identification of potential determinants (enzymes, transporters or transcription factors) and regulatory mechanisms responsible for high-quality oil accumulation. RESULTS: To select superior germplasm and unravel regulatory mechanism of high oil production for developing L. glauca fruit oils as biodiesel, 8 plus trees (accession LG01/02/03/04/05/06/07/08) with high-yield fruits were selected to evaluate the differences in oil content, FA profile, biodiesel yield and fuel property, and to construct fuel property prediction model, revealing a variation in the levels of fruit oil (45.12-60.95%), monounsaturated FA (52.43-78.46%) and polyunsaturated FA (17.69-38.73%), and biodiesel yield (80.12-98.71%) across different accessions. Of note, LG06 had a maximum yield of oil (60.95%) and biodiesel (98.71%), and ideal proportions of C18:1 (77.89%), C18:2 (14.16%) and C18:3 (1.55%), indicating that fruit oils from accession LG06 was the most suitable for high-quality biodiesel production. To highlight molecular mechanism that govern such differences in oil content and FA composition of different accessions, the quantitative relationship between oil-synthesized gene transcription and oil accumulative amount were conducted on different accessions to identify some vital determinants (enzymes, transporters or transcription factors) with a model of carbon metabolic regulatory for high-quality oil accumulation by an integrated analysis of our recent transcriptome data and qRT-PCR detection. Our findings may present strategies for developing L. glauca fruit oils as biodiesel feedstock and engineering its oil accumulation. CONCLUSIONS: This is the first report on the cross-accessions evaluations of L. glauca fruit oils to determine ideal accession for producing ideal biodiesel, and the associations of oil accumulative amount with oil-synthesized gene transcription was performed to identify some crucial determinants (enzymes, transporters or transcription factors) with metabolic regulation model established for governing high oil production. Our finding may provide molecular basis for new strategies of developing biodiesel resource and engineering oil accumulation.

Transcriptome analysis of genes involved in starch biosynthesis in developing Chinese chestnut (Castanea mollissima Blume) seed kernels.

Chinese chestnut (Castanea mollissima Blume) seed kernels (CCSK) with high quality and quantity of starch has emerged as a potential raw material for food industry, but the molecular regulatory mechanism of starch accumulation in developing CCSK is still unclear. In this study, we firstly analyzed the fruit development, starch accumulation, and microscopic observation of dynamic accumulation of starch granules of developing CCSK from 10 days after flowering (DAF) to 100 DAF, of which six representative CCSK samples (50-100 DAF) were selected for transcriptome sequencing analysis. Approximately 40 million valid reads were obtained, with an average length of 124.95 bp, which were searched against a reference genome, returning 38,146 unigenes (mean size = 1164.19 bp). Using the DESeq method, 1968, 1573, 1187, 1274, and 1494 differentially expressed unigenes were identified at 60:50, 70:60, 80:70, 90:80 and 100:90 DAF, respectively. The relationship between the unigene transcriptional profiles and starch dynamic patterns in developing CCSK was comparatively analyzed, and the specific unigenes encoding for metabolic enzymes (SUSY2, PGM, PGI, GPT, NTT, AGP3, AGP2, GBSS1, SS1, SBE1, SBE2.1, SBE2.2, ISA1, ISA2, ISA3, and PHO) were characterized to be involved potentially in the biosynthesis of G-1-P, ADPG, and starch. Finally, the temporal transcript profiles of genes encoding key enzymes (susy2, pgi2, gpt1, agp2, agp3, gbss1, ss1, sbe1, sbe2.1, sbe2.2, isa1, isa2, isa3, and pho) were validated by quantitative real-time PCR (qRT-PCR). Our findings could help to reveal the molecular regulatory mechanism of starch accumulation in developing CCSK and may also provide potential candidate genes for increasing starch content in Chinese chestnut or other starchy crops.

Populus euphratica remorin 6.5 activates plasma membrane H+-ATPases to mediate salt tolerance.

Remorins (REMs) play an important role in the ability of plants to adapt to adverse environments. PeREM6.5, a protein of the REM family in Populus euphratica (salt-resistant poplar), was induced by NaCl stress in callus, roots and leaves. We cloned the full-length PeREM6.5 from P. euphratica and transformed it into Escherichia coli and Arabidopsis thaliana. PeREM6.5 recombinant protein significantly increased the H+-ATPase hydrolytic activity and H+ transport activity in P. euphratica plasma membrane (PM) vesicles. Yeast two-hybrid assay showed that P. euphratica REM6.5 interacted with RPM1-interacting protein 4 (PeRIN4). Notably, the PeREM6.5-induced increase in PM H+-ATPase activity was enhanced by PeRIN4 recombinant protein. Overexpression of PeREM6.5 in Arabidopsis significantly improved salt tolerance in transgenic plants in terms of survival rate, root growth, electrolyte leakage and malondialdehyde content. Arabidopsis plants overexpressing PeREM6.5 retained high PM H+-ATPase activity in both in vivo and in vitro assays. PeREM6.5-transgenic plants had reduced accumulation of Na+ due to the Na+ extrusion promoted by the H+-ATPases. Moreover, the H+ pumps caused hyperpolarization of the PM, which reduced the K+ loss mediated by the depolarization-activated channels in the PM of salinized roots. Therefore, we conclude that PeREM6.5 regulated H+-ATPase activity in the PM, thus enhancing the plant capacity to maintain ionic homeostasis under salinity.

Populus euphratica WRKY1 binds the promoter of H+-ATPase gene to enhance gene expression and salt tolerance.

Plasma membrane proton pumps play a crucial role in maintaining ionic homeostasis in salt-resistant Populus euphratica under saline conditions. High levels of NaCl (200 mM) induced PeHA1 expression in P. euphratica roots and leaves. We isolated a 2022 bp promoter fragment upstream of the translational start of PeHA1 from P. euphratica. The promoter-reporter construct PeHA1-pro::GUS was transferred to tobacco plants, demonstrating that ß-glucuronidase activities increased in root, leaf, and stem tissues under salt stress. DNA affinity purification sequencing revealed that PeWRKY1 protein targeted the PeHA1 gene. We assessed the salt-induced transcriptional response of PeWRKY1 and its interaction with PeHA1 in P. euphratica. PeWRKY1 binding to the PeHA1 W-box in the promoter region was verified by a yeast one-hybrid assay, EMSA, luciferase reporter assay, and virus-induced gene silencing. Transgenic tobacco plants overexpressing PeWRKY1 had improved expression of NtHA4, which has a cis-acting W-box in the regulatory region, and improved H+ pumping activity in both in vivo and in vitro assays. We conclude that salt stress up-regulated PeHA1 transcription due to the binding of PeWRKY1 to the W-box in the promoter region of PeHA1. Thus, we conclude that enhanced H+ pumping activity enabled salt-stressed plants to retain Na+ homeostasis.

Populus euphratica JRL Mediates ABA Response, Ionic and ROS Homeostasis in Arabidopsis under Salt Stress.

Sodium chloride (NaCl) induced expression of a jacalin-related mannose-binding lectin (JRL) gene in leaves, roots, and callus cultures of Populus euphratica (salt-resistant poplar). To explore the mechanism of the PeJRL in salinity tolerance, the full length of PeJRL was cloned from P. euphratica and was transformed into Arabidopsis. PeJRL was localized to the cytoplasm in mesophyll cells. Overexpression of PeJRL in Arabidopsis significantly improved the salt tolerance of transgenic plants, in terms of seed germination, root growth, and electrolyte leakage during seedling establishment. Under NaCl stress, transgenic plants retained K⁺ and limited the accumulation of Na⁺. PeJRL-transgenic lines increased Na⁺ extrusion, which was associated with the upward regulation of SOS1, AHA1, and AHA2 genes encoding plasma membrane Na⁺/proton (H⁺) antiporter and H⁺-pumps. The activated H⁺-ATPases in PeJRL-overexpressed plants restricted the channel-mediated loss of K⁺ that was activated by NaCl-induced depolarization. Under salt stress, PeJRL⁻transgenic Arabidopsis maintained reactive oxygen species (ROS) homeostasis by activating the antioxidant enzymes and reducing the production of O2- through downregulation of NADPH oxidases. Of note, the PeJRL-transgenic Arabidopsis repressed abscisic acid (ABA) biosynthesis, thus reducing the ABA-elicited ROS production and the oxidative damage during the period of salt stress. A schematic model was proposed to show the mediation of PeJRL on ABA response, and ionic and ROS homeostasis under NaCl stress.

Amelioration of nitrate uptake under salt stress by ectomycorrhiza with and without a Hartig net.

Salt stress is an important environmental cue impeding poplar nitrogen nutrition. Here, we characterized the impact of salinity on proton-driven nitrate fluxes in ectomycorrhizal roots and the importance of a Hartig net for nitrate uptake. We employed two Paxillus involutus strains for root colonization: MAJ, which forms typical ectomycorrhizal structures (mantle and Hartig net), and NAU, colonizing roots with a thin, loose hyphal sheath. Fungus-colonized and noncolonized Populus × canescens were exposed to sodium chloride and used to measure root surface pH, nitrate (NO3- ) flux and transcription of NO3- transporters (NRTs; PcNRT1.1, -1.2, -2.1), and plasmalemma proton ATPases (HAs; PcHA4, -8, -11). Paxillus colonization enhanced root NO3- uptake, decreased surface pH, and stimulated NRTs and HA4 of the host regardless the presence or absence of a Hartig net. Under salt stress, noncolonized roots exhibited strong net NO3- efflux, whereas beneficial effects of fungal colonization on surface pH and HAs prevented NO3- loss. Inhibition of HAs abolished NO3- influx under all conditions. We found that stimulation of HAs was crucial for the beneficial influence of ectomycorrhiza on NO3- uptake, whereas the presence of a Hartig net was not required for improved NO3- translocation. Mycorrhizas may contribute to host adaptation to salt-affected environments by keeping up NO3- nutrition.

Comprehensive evaluation of fuel properties and complex regulation of intracellular transporters for high oil production in developing seeds of Prunus sibirica for woody biodiesel.

BACKGROUND: Based on our previous studies of 17 Prunus sibirica germplasms, one plus tree with high quality and quantity of seed oils has emerged as novel potential source of biodiesel. To better develop P. sibirica seed oils as woody biodiesel, a concurrent exploration of oil content, FA composition, biodiesel yield and fuel properties as well as prediction model construction for fuel properties was conducted on developing seeds to determine the optimal seed harvest time for producing high-quality biodiesel. Oil synthesis required supply of carbon source, energy and FA, but their transport mechanisms still remains enigmatic. Our recent 454 sequencing of P. sibirica could provide long-read sequences to identify membrane transporters for a better understanding of regulatory mechanism for high oil production in developing seeds. RESULTS: To better develop the seed oils of P. sibirica as woody biodiesel, we firstly focused on a temporal and comparative evaluation of growth tendency, oil content, FA composition, biodiesel yield and fuel properties as well as model construction for biodiesel property prediction in different developing seeds from P. sibirica plus tree (accession AS-80), revealing that the oils from developing seeds harvested after 60 days after flowering (DAF) could be as novel potential feedstock for producing biodiesel with ideal fuel property. To gain new insight into membrane transport mechanism for high oil yield in developing seeds of P. sibirica, we presented a global analysis of transporter based on our recent 454 sequencing data of P. sibirica. We annotated a total of 116 genes for membrane-localized transporters at different organelles (plastid, endoplasmatic reticulum, tonoplast, mitochondria and peroxisome), of which some specific transporters were identified to be involved in carbon allocation, metabolite transport and energy supply for oil synthesis by both RT-PCR and qRT-PCR. Importantly, the transporter-mediated model was well established for high oil synthesis in developing P. sibirica seeds. Our findings could help to reveal molecular mechanism of increased oil production and may also present strategies for engineering oil accumulation in oilseed plants. CONCLUSIONS: This study presents a temporal and comparative evaluation of developing P. sibirica seed oils as a potential feedstock for producing high-quality biodiesel and a global identification for membrane transporters was to gain better insights into regulatory mechanism of high oil production in developing seeds of P. sibirica. Our findings may present strategies for developing woody biodiesel resources and engineering oil accumulation.

The complete plastid genome of Chinese cinnamon, Cinnamomum aromaticum Nees (Lauraceae).

Cinnamomum aromaticum has long been recognized and cultivated in tropical and subtropical Asia for their aromatic bark to produce cinnamon. We reported for the first time the complete plastid genome of C. aromaticum and reconstructed its phylogenetic position. The complete plastid genome is 152,754 bp in length with a quadripartite organization: a large single copy (LSC) region of 93,706 bp and a small single copy (SSC) region of 18,916 bp. Each of the two inverted repeat regions (IRa and IRb) is 20,066 bp. We recovered 128 functional genes, including 84 protein-coding genes, 36 tRNA genes and 8 rRNA genes. The phylogenetic analysis suggested that C. aromaticum and two samples of C. camphora forms a strongly supported clade, which is sister to another cinnamon species of C. verum native to Sri Lanka with strong ultrafast bootstrap support.

Hydrogen Sulfide Mediates K+ and Na+ Homeostasis in the Roots of Salt-Resistant and Salt-Sensitive Poplar Species Subjected to NaCl Stress.

Non-invasive micro-test techniques (NMT) were used to analyze NaCl-altered flux profiles of K+, Na+, and H+ in roots and effects of NaHS (a H2S donor) on root ion fluxes in two contrasting poplar species, Populus euphratica (salt-resistant) and Populus popularis (salt-sensitive). Both poplar species displayed a net K+ efflux after exposure to salt shock (100 mM NaCl), as well as after short-term (24 h), and long-term (LT) (5 days) saline treatment (50 mM NaCl, referred to as salt stress). NaHS (50 µM) restricted NaCl-induced K+ efflux in roots irrespective of the duration of salt exposure, but K+ efflux was not pronounced in data collected from the LT salt stress treatment of P. euphratica. The NaCl-induced K+ efflux was inhibited by a K+ channel blocker, tetraethylammonium chloride (TEA) in P. popularis root samples, but K+ loss increased with a specific inhibitor of plasma membrane (PM) H+-ATPase, sodium orthovanadate, in both poplar species under LT salt stress and NaHS treatment. This indicates that NaCl-induced K+ loss was through depolarization-activated K+ channels. NaHS caused increased Na+ efflux and a corresponding increase in H+ influx for poplar roots subjected to both the short- and LT salt stress. The NaHS-enhanced H+ influx was not significant in P. euphratica samples subjected to short term salt stress. Both sodium orthovanadate and amiloride (a Na+/H+ antiporter inhibitor) effectively inhibited the NaHS-augmented Na+ efflux, indicating that the H2S-enhanced Na+ efflux was due to active Na+ exclusion across the PM. We therefore conclude that the beneficial effects of H2S probably arise from upward regulation of the Na+/H+ antiport system (H+ pumps and Na+/H+ antiporters), which promote exchange of Na+ with H+ across the PM and simultaneously restricted the channel-mediated K+ loss that activated by membrane depolarization.

Identification of AUXIN RESPONSE FACTOR gene family from Prunus sibirica and its expression analysis during mesocarp and kernel development.

BACKGROUND: Auxin response factors (ARFs) in auxin signaling pathway are an important component that can regulate the transcription of auxin-responsive genes involved in almost all aspects of plant growth and development. To our knowledge, the comprehensive and systematic characterization of ARF genes has never been reported in Prunus sibirica, a novel woody biodiesel feedstock in China. RESULTS: In this study, we identified 14 PsARF genes with a perfect open reading frame (ORF) in P. sibirica by using its previous transcriptomic data. Conserved motif analysis showed that all identified PsARF proteins had typical DNA-binding and ARF domain, but 5 members (PsARF3, 8 10, 16 and 17) lacked the dimerization domain. Phylogenetic analysis of the ARF proteins generated from various plant species indicated that ARFs could be categorized into 4 major groups (Class I, II, III and IV), in which all identified ARFs from P. sibirica showed a closest relationship with those from P. mume. Comparison of the expression profiles of 14 PsARF genes in different developmental stages of Siberian apricot mesocarp (SAM) and kernel (SAK) reflected distinct temporal or spatial expression patterns for PsARF genes. Additionally, based on the expressed data from fruit and seed development of multiple plant species, we identified 1514 ARF-correlated genes using weighted gene co-expression network analysis (WGCNA). And the major portion of ARF-correlated gene was characterized to be involved in protein, nucleic acid and carbohydrate metabolic, transport and regulatory processes. CONCLUSIONS: In summary, we systematically and comprehensively analyzed the structure, expression pattern and co-expression network of ARF gene family in P. sibirica. All our findings provide theoretical foundation for the PsARF gene family and will pave the way for elucidating the precise role of PsARF genes in SAM and SAK development.

Salt-Sensitive Signaling Networks in the Mediation of K+/Na+ Homeostasis Gene Expression in Glycyrrhiza uralensis Roots.

We investigated the effects of salt-sensitive signaling molecules on ionic fluxes and gene expression related to K+/Na+ homeostasis in a perennial herb, Glycyrrhiza uralensis, during short-term NaCl stress (100 mM, 24 h). Salt treatment caused more pronounced Na+ accumulation in root cells than in leaf cells. Na+ ions were mostly compartmentalized in vacuoles. Roots exposed to NaCl showed increased levels of extracellular ATP (eATP), cytosolic Ca2+, H2O2, and NO. Steady-state flux recordings revealed that these salt-sensitive signaling molecules enhanced NaCl-responsive Na+ efflux, due to the activated Na+/H+ antiport system in the plasma membrane (PM). Moreover, salt-elicited K+ efflux, which was mediated by depolarization-activated cation channels, was reduced with the addition of Ca2+, H2O2, NO, and eATP. The salt-adaptive effects of these molecules (Na+ extrusion and K+ maintenance) were reduced by pharmacological agents, including LaCl3 (a PM Ca2+ channel inhibitor), DMTU (a reactive oxygen species scavenger), cPTIO (an NO scavenger), or PPADS (an antagonist of animal PM purine P2 receptors). RT-qPCR data showed that the activation of the PM Na+/H+ antiport system in salinized roots most likely resulted from the upregulation of two genes, GuSOS1 and GuAHA, which encoded the PM Na+/H+ antiporter, salt overly sensitive 1 (SOS1), and H+-ATPase, respectively. Clear interactions occurred between these salt-sensitive agonists to accelerate transcription of salt-responsive signaling pathway genes in G. uralensis roots. For example, Ca2+, H2O2, NO, and eATP promoted transcription of GuSOS3 (salt overly sensitive 3) and/or GuCIPK (CBL-interacting protein kinase) to activate the predominant Ca2+-SOS signaling pathway in salinized liquorice roots. eATP, a novel player in the salt response of G. uralensis, increased the transcription of GuSOS3, GuCIPK, GuRbohD (respiratory burst oxidase homolog protein D), GuNIR (nitrate reductase), GuMAPK3, and GuMAPK6 (the mitogen-activated protein kinases 3 and 6). Moreover, GuMAPK3 and GuMAPK6 expression levels were enhanced by H2O2 in NaCl-stressed G. uralensis roots. Our results indicated that eATP triggered downstream components and interacted with Ca2+, H2O2, and NO signaling to maintain K+/Na+ homeostasis. We propose that a multiple signaling network regulated K+/Na+ homeostasis in NaCl-stressed G. uralensis roots.

Integrated analysis of 454 and Illumina transcriptomic sequencing characterizes carbon flux and energy source for fatty acid synthesis in developing Lindera glauca fruits for woody biodiesel.

BACKGROUND: Lindera glauca fruit with high quality and quantity of oil has emerged as a novel potential source of biodiesel in China, but the molecular regulatory mechanism of carbon flux and energy source for oil biosynthesis in developing fruits is still unknown. To better develop fruit oils of L. glauca as woody biodiesel, a combination of two different sequencing platforms (454 and Illumina) and qRT-PCR analysis was used to define a minimal reference transcriptome of developing L. glauca fruits, and to construct carbon and energy metabolic model for regulation of carbon partitioning and energy supply for FA biosynthesis and oil accumulation. RESULTS: We first analyzed the dynamic patterns of growth tendency, oil content, FA compositions, biodiesel properties, and the contents of ATP and pyridine nucleotide of L. glauca fruits from seven different developing stages. Comprehensive characterization of transcriptome of the developing L. glauca fruit was performed using a combination of two different next-generation sequencing platforms, of which three representative fruit samples (50, 125, and 150 DAF) and one mixed sample from seven developing stages were selected for Illumina and 454 sequencing, respectively. The unigenes separately obtained from long and short reads (201, and 259, respectively, in total) were reconciled using TGICL software, resulting in a total of 60,031 unigenes (mean length = 1061.95 bp) to describe a transcriptome for developing L. glauca fruits. Notably, 198 genes were annotated for photosynthesis, sucrose cleavage, carbon allocation, metabolite transport, acetyl-CoA formation, oil synthesis, and energy metabolism, among which some specific transporters, transcription factors, and enzymes were identified to be implicated in carbon partitioning and energy source for oil synthesis by an integrated analysis of transcriptomic sequencing and qRT-PCR. Importantly, the carbon and energy metabolic model was well established for oil biosynthesis of developing L. glauca fruits, which could help to reveal the molecular regulatory mechanism of the increased oil production in developing fruits. CONCLUSIONS: This study presents for the first time the application of an integrated two different sequencing analyses (Illumina and 454) and qRT-PCR detection to define a minimal reference transcriptome for developing L. glauca fruits, and to elucidate the molecular regulatory mechanism of carbon flux control and energy provision for oil synthesis. Our results will provide a valuable resource for future fundamental and applied research on the woody biodiesel plants.

Integrated mRNA and miRNA transcriptome reveal a cross-talk between developing response and hormone signaling for the seed kernels of Siberian apricot.

Recently, our transcriptomic analysis has identified some functional genes responsible for oil biosynthesis in developing SASK, yet miRNA-mediated regulation for SASK development and oil accumulation is poorly understood. Here, 3 representative periods of 10, 30 and 60 DAF were selected for sRNA sequencing based on the dynamic patterns of growth tendency and oil content of developing SASK. By miRNA transcriptomic analysis, we characterized 296 known and 44 novel miRNAs in developing SASK, among which 36 known and 6 novel miRNAs respond specifically to developing SASK. Importantly, we performed an integrated analysis of mRNA and miRNA transcriptome as well as qRT-PCR detection to identify some key miRNAs and their targets (miR156-SPL, miR160-ARF18, miR164-NAC1, miR171h-SCL6, miR172-AP2, miR395-AUX22B, miR530-P2C37, miR393h-TIR1/AFB2 and psi-miRn5-SnRK2A) potentially involved in developing response and hormone signaling of SASK. Our results provide new insights into the important regulatory function of cross-talk between development response and hormone signaling for SASK oil accumulation.

Cross-talk between freezing response and signaling for regulatory transcriptions of MIR475b and its targets by miR475b promoter in Populus suaveolens.

MicroRNAs (miRNAs) are small, non-coding RNAs that play important roles in post-transcriptional regulation of their target genes, yet the transcriptional regulation of plant miRNAs by promoter is poorly understood. Here, we firstly clone pri-miR475b cDNA and its native promoter from P. suaveolens, and characterize Psu-MIR475b as class-II gene transcribed by RNA polymerase II. By 5' deletion analysis of Psu-miR475b promoter in a series of promoter-GUS chimeric vectors, we functionally identify three positive regulatory regions and multiple cis-acting elements responsible for Psu-miR475b promoter activity in response to freezing stress and exogenous hormone treatment. Moreover, the Psu-miR475b promoter activity displays a tissue-specific manner, negatively regulated by freezing stress and positively by MeJA, SA or GA treatment. Importantly, we comparatively analyze the time-course transcriptional profiles of Psu-miR475b and its targets in Psu-miR475b over-expression transgenic plants controlled by Psu-miR475b-specific promoter or CaMV 35S constitutive promoter, and explore the regulatory mechanism of Psu-miR475b promoter controlling transcriptional expressions of Psu-MIR475b and its targets in response to freezing stress and exogenous hormone treatment. Our results reveal that Psu-miR475b promoter-mediated transcriptions of Psu-MIR475b and its targets in response to freezing stress may be involved in a cross-talk between freezing response and stress signaling process.