A genome for Cissus illustrates features underlying its evolutionary success in dry savannas.

Cissus is the largest genus in Vitaceae and is mainly distributed in the tropics and subtropics. Crassulacean acid metabolism (CAM), a photosynthetic adaptation to the occurrence of succulent leaves or stems, indicates that convergent evolution occurred in response to drought stress during species radiation. Here we provide the chromosomal level assembly of Cissus rotundifolia (an endemic species in Eastern Africa) and a genome-wide comparison with grape to understand genome divergence within an ancient eudicot family. Extensive transcriptome data were produced to illustrate the genetics underpinning C. rotundifolia's ecological adaption to seasonal aridity. The modern karyotype and smaller genome of C. rotundifolia (n = 12, 350.69 Mb/1C), which lack further whole-genome duplication, were mainly derived from gross chromosomal rearrangements such as fusions and segmental duplications, and were sculpted by a very recent burst of retrotransposon activity. Bias in local gene amplification contributed to its remarkable functional divergence from grape, and the specific proliferated genes associated with abiotic and biotic responses (e.g. HSP-20, NBS-LRR) enabled C. rotundifolia to survive in a hostile environment. Reorganization of existing enzymes of CAM characterized as diurnal expression patterns of relevant genes further confer the ability to thrive in dry savannas.

Effect of Stray Current on Corrosion and Calcium Ion Corrosion of Concrete Reinforcement.

The construction of subways, hydroelectric stations and water substations is exposed to stray currents, which can accelerate concrete corrosion. The influence of stray currents on reinforced concrete structures is unclear. In this paper, the influence of concrete strength grade, reinforcement diameter and stray current intensity on the extent of reinforcement corrosion and calcium ion dissolution were investigated, and the damage of reinforcement and calcium ion corrosion to concrete strength was investigated by simulating a stray current environment and conducting an electrified acceleration test. The test results show that the higher the strength grade of concrete, the lower the stray current intensity and the smaller the corrosion rate and calcium ion dissolution rate of reinforcement; with the increase in the diameter of reinforcement the corrosion rate decreases, but the calcium ion dissolution rate is not affected by reinforcement diameter. The damage effect of reinforcement corrosion on concrete compressive strength is more obvious than that of calcium ion corrosion.

Perioperative Electroacupuncture Can Accelerate the Recovery of Gastrointestinal Function in Cancer Patients Undergoing Pancreatectomy or Gastrectomy: A Randomized Controlled Trial.

The effect of perioperative acupuncture on accelerating gastrointestinal function recovery has been reported in colorectal surgery and distal gastrectomy (Billroth-II). However, the evidence in pancreatectomy and other gastrectomy is still limited. A prospective, randomized controlled trial was conducted between May 2018 and August 2019. Consecutive patients undergoing pancreatectomy or gastrectomy in our hospital were randomly assigned to the electroacupuncture (EA) group and the control group. The patients in the EA group received transcutaneous EA on Bai-hui (GV20), Nei-guan (PC6), Tian-shu (ST25), and Zu-san-li (ST36) once a day in the afternoon, and the control group received sham EA. Primary outcomes were the time to first flatus and time to first defecation. In total, 461 patients were randomly assigned to the groups, and 385 were analyzed finally (EA group, n = 201; control group, n = 184). Time to first flatus (3.0 ± 0.7 vs 4.2 ± 1.0, P < 0.001) and first defecation (4.2 ± 0.9 vs 5.4 ± 1.2, P < 0.001) in the EA group were significantly shorter than those in the control group. Of patients undergoing pancreatectomy, those undergoing pancreaticoduodenectomy and intraoperative radiation therapy (IORT) surgery benefitted from EA in time to first flatus (P < 0.001) and first defecation (P < 0.001), while those undergoing distal pancreatectomy did not (P flatus=0.157, P defecation=0.007) completely. Of patients undergoing gastrectomy, those undergoing total gastrectomy and distal gastrectomy (Billroth-II) benefitted from EA (P < 0.001), as did those undergoing proximal gastrectomy (P=0.015). Patients undergoing distal gastrectomy (Billroth-I) benefitted from EA in time to first defecation (P=0.012) but not flatus (P=0.051). The time of parenteral nutrition, hospital stay, and time to first independent walk in the EA group were shorter than those in the control group. No severe EA complications were reported. EA was safe and effective in accelerating postoperative gastrointestinal function recovery. Patients undergoing pancreaticoduodenectomy, IORT surgery, total gastrectomy, proximal gastrectomy, or distal gastrectomy (Billroth-II) could benefit from EA. This trial is registered with NCT03291574.

[XUE Li-gong's clinical thinking of "unknotting" method with long-round needle in treatment of painful bi syndrome of meridian muscle region].

Professor XUE Li-gong's clinical experiences were summarized in treatment of painful bi syndrome of meridian muscle region with the "unknotting" method of long-round needle. It is believed that painful bi syndrome of meridian muscle region is related chiefly with the invasion of wind, cold and damp pathogens, exertion and traumatic injury. These pathogenic factors induce the "transverse-collateral" entrapment in the local and result in refractory painful bi syndrome of meridian muscle region. The "unknotting" method is adopted with long-round needle, which can either separate bluntly the knotted lesions or cut them sharply. "Taking the painful sites as the points" is the principle of point selection in treatment of meridian muscle disorder. Regarding needling techniques, joint needling, lateral needling and short needling are predominated.

Diurnal Regulation of Plant Epidermal Wax Synthesis through Antagonistic Roles of the Transcription Factors SPL9 and DEWAX.

Plant surface waxes form an outer barrier that protects the plant from many forms of environmental stress. The deposition of cuticular waxes on the plant surface is regulated by external environmental changes, including light and dark cycles. However, the underlying molecular mechanisms controlling light regulation of wax production are still poorly understood, especially at the posttranscriptional level. In this paper, we report the regulation of cuticular wax production by the miR156-SPL9 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9) module in Arabidopsis (Arabidopsis thaliana). When compared with wild-type plants, miR156 and SPL9 mutants showed significantly altered cuticular wax amounts in both stems and leaves. Furthermore, it was found that SPL9 positively regulates gene expression of the alkane-forming enzyme ECERIFERUM1 (CER1), as well as the primary (1-) alcohol-forming enzyme ECERIFERUM4 (CER4), to enhance alkane and 1-alcohol synthesis, respectively. Our results indicate that complex formation of SPL9 with a negative regulator of wax synthesis, DEWAX, will hamper SPL9 DNA binding ability, possibly by interfering with SPL9 homodimerization. Combined with their diurnal gene and protein expressions, this dynamic repression-activation transcriptional module defines a dynamic mechanism that may allow plants to optimize wax synthesis during daily cycles. These findings provide a regulatory framework for environmental signal integration in the regulation of wax synthesis.

Tung Tree (Vernicia fordii, Hemsl.) Genome and Transcriptome Sequencing Reveals Co-Ordinate Up-Regulation of Fatty Acid ß-Oxidation and Triacylglycerol Biosynthesis Pathways During Eleostearic Acid Accumulation in Seeds.

The tung tree (Vernicia fordii) is one of only a few plant species that produces high oil-yielding seeds rich in α-eleostearic acid (α-ESA, 18:3Δ9cis, 11trans, 13trans), a conjugated trienoic fatty acid with valuable industrial and medical properties. Previous attempts have been made to engineer tung oil biosynthesis in transgenic oilseed crops, but these efforts have met with limited success. Here we present a high-quality genome assembly and developing seed transcriptomic data set for this species. Whole-genome shotgun sequencing generated 176 Gb of genome sequence data used to create a final assembled sequence 1,176,320 kb in size, with a scaffold N50 size of >474 kb, and containing approximately 47,000 protein-coding genes. Genomic and transcriptomic data revealed full-length candidate genes for most of the known and suspected reactions that are necessary for fatty acid desaturation/conjugation, acyl editing and triacylglycerol biosynthesis. Seed transcriptomic analyses also revealed features unique to tung tree, including unusual transcriptional profiles of fatty acid biosynthetic genes, and co-ordinated (and seemingly paradoxical) simultaneous up-regulation of both fatty acid ß-oxidation and triacylglycerol biosynthesis in mid-development seeds. The precise temporal control of the expression patterns for these two pathways may account for α-ESA enrichment in tung seeds, while controlling the levels of potentially toxic by-products. Deeper understanding of these processes may open doors to the design of engineered oilseeds containing high levels of α-ESA.

Potential of rhBMP-2 and dexamethasone-loaded Zein/PLLA scaffolds for enhanced in vitro osteogenesis of mesenchymal stem cells.

Nanofibers fabricated by electrospinning simulate the extracellular matrix of bone cells and so researchers have taken a keen interest in them for regenerating bone tissue. The aim of this study was to fabricate ideal Zein/PLLA nanofibers by coaxial electrospinning and to load them with bone morphogenetic protein 2 (BMP-2) and dexamethasone (DEX) for dual controlled-release for bone tissue engineering applications. Morphology, surface hydrophilicity and core-shell construction were analyzed by environmental scanning electron microscopy (SEM), water contact angle and transmission electron microscopy (TEM). The properties of the scaffolds were studied in terms of the viability, morphology and osteogenic differentiation of mesenchymal stem cells (MSCs) that had been cultured on nanofiber mats of the Zein/PLLA and were determined using SEM, CCK-8 assay, quantitative ALP staining analysis, quantitative mineral deposition using Alizarin red staining (ARS), immunofluorescence staining and western blot analysis of osteogenic proteins. In vitro studies demonstrated that the biological activity of DEX and BMP-2 was retained in the dual-drug-loaded nanofiber scaffolds. A large quantity of DEX was released in the first three days, while the release of BMP-2 lasted for more than 21 days. In vitro osteogenesis studies showed that the drug-loaded nanofiber scaffolds induced osteogenic differentiation. Furthermore, the dual controlled-release of BMP-2 and DEX enhanced the osteogenic differentiation of MSCs resulting from synergistic effects. Therefore, Zein/PLLA nanofiber scaffolds loaded with BMP-2 and DEX have great potential in bone tissue engineering applications.

RhBMP-2-loaded Poly(lactic-co-glycolic acid) microspheres fabricated by coaxial electrospraying for protein delivery.

In this study, we fabricated recombinant human bone morphogenetic protein-2 (rhBMP-2) loaded Poly(lactic-co-glycolic acid) (PLGA) microspheres with core-shell structures and particle sizes ranging from 2.5 to 8 µm by coaxial electrospraying. The manufacturing process of core-shell microspheres by coaxial electrospraying is simpler than that with other methods, and a smaller diameter can be obtained. The microspheres were analyzed by environmental scanning electron microscopy, transmission electron microscopy (TEM), and laser scanning confocal microscopy (LSCM). Moreover, the drug release profiles and degradation of rhBMP-2-loaded PLGA microspheres in vitro were investigated for 21 days and for 7 weeks, respectively. The rhBMP-2 was stabilized by using bovine serum albumin (BSA) to ensure protein activity in the electrospraying process. Fluorescently labeled protein that was loaded into the core-shell PLGA microspheres was verified by LSCM. The distinct layered structure that existed in the manufactured core-shell microspheres can be observed by TEM. Cell Counting Kit-8 (CCK-8) indicated that the core-shell PLGA microspheres loaded with rhBMP-2 have great potential for the treatment of bone defects, for bone regeneration, and in bone tissue engineering.

The Arabidopsis endoplasmic reticulum associated degradation pathways are involved in the regulation of heat stress response.

The Cytosolic Protein Response (CPR) in the cytosol and the Unfolded Protein Response (UPR) and ER-associated degradation (ERAD) in the endoplasmic reticulum are major pathways of the cellular proteostasis network. However, despite years of effort, how these protein quality control systems coordinated in vivo remains largely unknown, particularly in plants. In this study, the roles of two evolutionarily conserved ERAD pathways (DOA10 and HRD1) in heat stress response were investigated through reverse genetic approaches in Arabidopsis. Phenotypic analysis of the mutants showed that the two ERAD pathways additively play negative roles in heat tolerance, which was demonstrated by higher survival rate and lower electrolyte leakage in the loss of function mutants compared to the wild type plants. Importantly, gene expression analysis revealed that the mutant plants showed elevated transcriptional regulation of several downstream genes, including those encoding CPR and UPR marker genes, under both basal and heat stress conditions. Finally, multiple components of ERAD genes exhibited rapid response to increasing temperature. Taken together, our data not only unravels key insights into the crosstalk between different protein quality control processes, but also provides candidate genes to genetically improve plant heat tolerance in the future.

The Acyl Desaturase CER17 Is Involved in Producing Wax Unsaturated Primary Alcohols and Cutin Monomers.

We report n-6 monounsaturated primary alcohols (C26:1, C28:1, and C30:1 homologs) in the cuticular waxes of Arabidopsis (Arabidopsis thaliana) inflorescence stem, a class of wax not previously reported in Arabidopsis. The Arabidopsis cer17 mutant was completely deficient in these monounsaturated alcohols, and CER17 was found to encode a predicted ACYL-COENZYME A DESATURASE LIKE4 (ADS4). Studies of the Arabidopsis cer4 mutant and yeast variously expressing CER4 (a predicted fatty acyl-CoA reductase) with CER17/ADS4, demonstrated CER4's principal role in synthesis of these monounsaturated alcohols. Besides unsaturated alcohol deficiency, cer17 mutants exhibited a thickened and irregular cuticle ultrastructure and increased amounts of cutin monomers. Although unsaturated alcohols were absent throughout the cer17 stem, the mutation's effects on cutin monomers and cuticle ultrastructure were much more severe in distal than basal stems, consistent with observations that the CER17/ADS4 transcript was much more abundant in distal than basal stems. Furthermore, distal but not basal stems of a double mutant deficient for both CER17/ADS4 and LONG-CHAIN ACYL-COA SYNTHETASE1 produced even more cutin monomers and a thicker and more disorganized cuticle ultrastructure and higher cuticle permeability than observed for wild type or either mutant parent, indicating a dramatic genetic interaction on conversion of very long chain acyl-CoA precursors. These results provide evidence that CER17/ADS4 performs n-6 desaturation of very long chain acyl-CoAs in both distal and basal stems and has a major function associated with governing cutin monomer amounts primarily in the distal segments of the inflorescence stem.

[Explanation of Evidence-based Guidelines of Clinical Practice with Acupuncture and Moxibustion: Adult Bronchial Asthma].

The development and compilation of Evidence-based Guidelines of Clinical Practice with Acupuncture and Moxibustion: Adult Bronchial Asthma are introduced from three aspects, named the guideline methodology, the guideline structure and the guideline content. Based on the acupuncture-moxibustion practice and clinical research, the evidence-based medicine method is adopted. During the development and compilation of the guideline, the characteristics and advantages of acupuncture and moxibustion are specially considered in the treatment of this disease; the latest optimum evidences at home and abroad, experts' experience and patients' value are closely integrated with each other. Additionally, the worldwide accepted assessments of evidence quality and the recommendation (GRADE system) are combined with the clinical evidences of the ancient and modern famous acupuncture-moxibustion experts, and the clinical research evidences are with the experts' consensus to the large extent. The purpose of the guideline is to provide the maximal guidance to the clinical physicians.

De novo Assembly and Characterization of the Fruit Transcriptome of Idesia polycarpa Reveals Candidate Genes for Lipid Biosynthesis.

Idesia polycarpa, is a valuable oilseed-producing tree of the Flacourtiaceae family that has the potential to fulfill edible oil production and is also a possible biofuel feedstock. The fruit is unique in that it contains both saturated and unsaturated lipids present in pericarp and seed, respectively. However, triglyceride synthesis and storage in tissues outside of the seeds has been poorly studied in previous researches. To gain insight into the unique properties of I. polycarpa fruit lipid synthesis, biochemical, and transcriptomic approaches were used to compare the lipid accumulation between pericarp and seed of the fruit. Lipid accumulation rates, final lipid content and composition were significantly different between two tissues. Furthermore, we described the annotated transcriptome assembly and differential gene expression analysis generated from the pericarp and seed tissues. The data allowed the identification of distinct candidate genes and reconstruction of lipid pathways, which may explain the differences of oil synthesis between the two tissues. The results may be useful for engineering alternative pathways for lipid production in non-seed or vegetative tissues.

[Interpretation of Evidence-based Guidelines of Clinical Practice with Acupuncture and Moxibustion:Migraine of the version 2014].

Evidence-based Guidelines of Clinical Practice with Acupuncture and Moxibustion:Migraine (Guideline) was updated in 2014 on the basis of the version 2011. In Guideline of the updated version, the evidence quality grade and GRADE were adopted for the evidence evaluation and recommendation grading in methodology, and the clinical feasibility was stressed. In text, the recognition of modern medicine was updated, the sta-ging treatment of acupuncture and moxibustion was emphasized, the inclusive articles were expanded and the important indexes of efficacy evaluation were increased and highlighted. The paper aims to provide the instruction for the application of Guideline of the version 2014.

Natural variation in ARF18 gene simultaneously affects seed weight and silique length in polyploid rapeseed.

Seed weight (SW), which is one of the three major factors influencing grain yield, has been widely accepted as a complex trait that is controlled by polygenes, particularly in polyploid crops. Brassica napus L., which is the second leading crop source for vegetable oil around the world, is a tetraploid (4×) species. In the present study, we identified a major quantitative trait locus (QTL) on chromosome A9 of rapeseed in which the genes for SW and silique length (SL) were colocated. By fine mapping and association analysis, we uncovered a 165-bp deletion in the auxin-response factor 18 (ARF18) gene associated with increased SW and SL. ARF18 encodes an auxin-response factor and shows inhibitory activity on downstream auxin genes. This 55-aa deletion prevents ARF18 from forming homodimers, in turn resulting in the loss of binding activity. Furthermore, reciprocal crossing has shown that this QTL affects SW by maternal effects. Transcription analysis has shown that ARF18 regulates cell growth in the silique wall by acting via an auxin-response pathway. Together, our results suggest that ARF18 regulates silique wall development and determines SW via maternal regulation. In addition, our study reveals the first (to our knowledge) QTL in rapeseed and may provide insights into gene cloning involving polyploid crops.

Salt stress reduces root meristem size by nitric oxide-mediated modulation of auxin accumulation and signaling in Arabidopsis.

The development of the plant root system is highly plastic, which allows the plant to adapt to various environmental stresses. Salt stress inhibits root elongation by reducing the size of the root meristem. However, the mechanism underlying this process remains unclear. In this study, we explored whether and how auxin and nitric oxide (NO) are involved in salt-mediated inhibition of root meristem growth in Arabidopsis (Arabidopsis thaliana) using physiological, pharmacological, and genetic approaches. We found that salt stress significantly reduced root meristem size by down-regulating the expression of PINFORMED (PIN) genes, thereby reducing auxin levels. In addition, salt stress promoted AUXIN RESISTANT3 (AXR3)/INDOLE-3-ACETIC ACID17 (IAA17) stabilization, which repressed auxin signaling during this process. Furthermore, salt stress stimulated NO accumulation, whereas blocking NO production with the inhibitor N(ω)-nitro-l-arginine-methylester compromised the salt-mediated reduction of root meristem size, PIN down-regulation, and stabilization of AXR3/IAA17, indicating that NO is involved in salt-mediated inhibition of root meristem growth. Taken together, these findings suggest that salt stress inhibits root meristem growth by repressing PIN expression (thereby reducing auxin levels) and stabilizing IAA17 (thereby repressing auxin signaling) via increasing NO levels.

The Putative E3 Ubiquitin Ligase ECERIFERUM9 Regulates Abscisic Acid Biosynthesis and Response during Seed Germination and Postgermination Growth in Arabidopsis.

The ECERIFERUM9 (CER9) gene encodes a putative E3 ubiquitin ligase that functions in cuticle biosynthesis and the maintenance of plant water status. Here, we found that CER9 is also involved in abscisic acid (ABA) signaling in seeds and young seedlings of Arabidopsis (Arabidopsis thaliana). The germinated embryos of the mutants exhibited enhanced sensitivity to ABA during the transition from reversible dormancy to determinate seedling growth. Expression of the CER9 gene is closely related to ABA levels and displays a similar pattern to that of ABSCISIC ACID-INSENSITIVE5 (ABI5), which encodes a positive regulator of ABA responses in seeds. cer9 mutant seeds exhibited delayed germination that is independent of seed coat permeability. Quantitative proteomic analyses showed that cer9 seeds had a protein profile similar to that of the wild type treated with ABA. Transcriptomics analyses revealed that genes involved in ABA biosynthesis or signaling pathways were differentially regulated in cer9 seeds. Consistent with this, high levels of ABA were detected in dry seeds of cer9. Blocking ABA biosynthesis by fluridone treatment or by combining an ABA-deficient mutation with cer9 attenuated the phenotypes of cer9. Whereas introduction of the abi1-1, abi3-1, or abi4-103 mutation could completely eliminate the ABA hypersensitivity of cer9, introduction of abi5 resulted only in partial suppression. These results indicate that CER9 is a novel negative regulator of ABA biosynthesis and the ABA signaling pathway during seed germination.

Cosuppression of RBCS3B in Arabidopsis leads to severe photoinhibition caused by ROS accumulation.

KEY MESSAGE: Cosuppression of an Arabidopsis Rubisco small subunit gene RBCS3B at Arabidopsis resulted in albino or pale green phenotypes which were caused by ROS accumulation As the most abundant protein on Earth, Rubisco has received much attention in the past decades. Even so, its function is still not understood thoroughly. In this paper, four Arabidopsis transgenic lines (RBCS3B-7, 18, 33, and 35) with albino or pale green phenotypes were obtained by transformation with a construct driving expression of sense RBCS3B, a Rubisco small subunit gene. The phenotypes produced in these transgenic lines were found to be caused by cosuppression. Among these lines, RBCS3B-7 displayed the most severe phenotypes including reduced height, developmental arrest and plant mortality before flowering when grown under normal light on soil. Chloroplast numbers in mesophyll cells were decreased compared to WT, and stacked thylakoids of chloroplasts were broken down gradually in RBCS3B-7 throughout development. In addition, the RBCS3B-7 line was light sensitive, and PSII activity measurement revealed that RBCS3B-7 suffered severe photoinhibition, even under normal light. We found that photoinhibition was due to accumulation of ROS, which accelerated photodamage of PSII and inhibited the repair of PSII in RBCS3B-7.

In vivo role of nitric oxide in plant response to abiotic and biotic stress.

Over the past few years, nitric oxide (NO) has emerged as an important regulator in many physiological events, especially in response to abiotic and biotic stress. However, the roles of NO were mostly derived from pharmacological studies or the mutants impaired NO synthesis unspecifically. In our recent study, we highlighted a novel strategy by expressing the rat neuronal NO synthase (nNOS) in Arabidopsis to explore the in vivo role of NO. Our results suggested that plants were able to perform well in the constitutive presence of nNOS, and provided a new class of plant experimental system with specific in vivo NO release. Furthermore, our findings also confirmed that the in vivo NO is essential for most of environmental abiotic stresses and disease resistance against pathogen infection. Proper level of NO may be necessary and beneficial, not only in plant response to the environmental abiotic stress, but also to biotic stress.

The BnGRF2 gene (GRF2-like gene from Brassica napus) enhances seed oil production through regulating cell number and plant photosynthesis.

Seed yield and oil content are two important agricultural characteristics in oil crop breeding, and a lot of functional gene research is being concentrated on increasing these factors. In this study, by differential gene expression analyses between rapeseed lines (zy036 and 51070) which exhibit different levels of seed oil production, BnGRF2 (Brassica napus growth-regulating factor 2-like gene) was identified in the high oil-producing line zy036. To elucidate the possible roles of BnGRF2 in seed oil production, the cDNA sequences of the rapeseed GRF2 gene were isolated. The Blastn result showed that rapeseed contained BnGRF2a/2b which were located in the A genome (A1 and A3) and C genome (C1 and C6), respectively, and the dominantly expressed gene BnGRF2a was chosen for transgenic research. Analysis of 35S-BnGRF2a transgenic Arabidopsis showed that overexpressed BnGRF2a resulted in an increase in seed oil production of >50%. Moreover, BnGRF2a also induced a >20% enlargement in extended leaves and >40% improvement in photosynthetic efficiency because of an increase in the chlorophyll content. Furthermore, transcriptome analyses indicated that some genes associated with cell proliferation, photosynthesis, and oil synthesis were up-regulated, which revealed that cell number and plant photosynthesis contributed to the increased seed weight and oil content. Because of less efficient self-fertilization induced by the longer pistil in the 35S-BnGRF2a transgenic line, Napin-BnGRF2a transgenic lines were further used to identify the function of BnGRF2, and the results showed that seed oil production also could increase >40% compared with the wild-type control. The results suggest that improvement to economically important characteristics in oil crops may be achieved by manipulation of the GRF2 expression level.

Increasing nitric oxide content in Arabidopsis thaliana by expressing rat neuronal nitric oxide synthase resulted in enhanced stress tolerance.

Nitric oxide (NO) plays essential roles in many physiological and developmental processes in plants, including biotic and abiotic stresses, which have adverse effects on agricultural production. However, due to the lack of findings regarding nitric oxide synthase (NOS), many difficulties arise in investigating the physiological roles of NO in vivo and thus its utilization for genetic engineering. Here, to explore the possibility of manipulating the endogenous NO level, rat neuronal NOS (nNOS) was expressed in Arabidopsis thaliana. The 35S::nNOS plants showed higher NOS activity and accumulation of NO using the fluorescent probe 3-amino, 4-aminomethyl-2', 7'-difluorescein, diacetate (DAF-FM DA) assay and the hemoglobin assay. Compared with the wild type, the 35S::nNOS plants displayed improved salt and drought tolerance, which was further confirmed by changes in physiological parameters including reduced water loss rate, reduced stomatal aperture, and altered proline and malondialdehyde content. Quantitative real-time PCR analyses revealed that the expression of several stress-regulated genes was up-regulated in the transgenic lines. Furthermore, the transgenic lines also showed enhanced disease resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 by activating the expression of defense-related genes. In addition, we found that the 35S::nNOS lines flowered late by regulating the expression of CO, FLC and LFY genes. Together, these results demonstrated that it is a useful strategy to exploit the roles of plant NO in various processes by the expression of rat nNOS. The approach may also be useful for genetic engineering of crops with increased environmental adaptations.