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We compared community composition and co-occurrence patterns of phyllosphere fungi between island and mainland populations within a single plant species (Mussaenda kwangtungensis) using high-throughput sequencing technology. We then used 11 microsatellite loci for host genotyping. The island populations differed significantly from their mainland counterparts in phyllosphere fungal community structure. Topological features of co-occurrence network showed geographic patterns wherein fungal assemblages were less complex, but more modular in island regions than mainland ones. Moreover, fungal interactions and community composition were strongly influenced by the genetic differentiation of host plants. This study may advance our understanding of assembly principles and ecological interactions of phyllosphere fungal communities, as well as improve our ability to optimize fungal utilization for the benefit of people.

Importation of taxadiene synthase into chloroplasts is important for the efficient heterologous production of taxadiene. Taxadiene, the first committed precursor to taxol, is synthesized from geranylgeranyl pyrophosphate (GGPP) by action of taxadiene synthase (TS). Heterologous production of taxadiene could potentially rely on both cytosolic mevalonic acid (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway. We suggest the compartmentalized engineering in chloroplast as an efficient approach for taxadiene production. In this study, we directly introduced the TS gene from Taxus brevifolia into the tobacco chloroplast genome and found that the transplastomic plants accumulated a low content of taxadiene, ~ 5.6 μg/g dry weight (DW). Moreover, we tried a combination of MEP and MVA pathways for taxadiene synthesis by nuclear transformation with a truncated version of TS (without encoding a transit peptide) into the transplastomic plants. However, this did not further improve the taxadiene production. In contrast, we found that taxadiene could be produced up to 87.8 μg/g DW in leaves of transgenic plants expressing TS with a chloroplast transit peptide, which was significantly higher than that in leaves of transplastomic plants. Thus, this study highlights the importance of TS importation into chloroplast for production of taxadiene.

Plastid transformation technology has several attractive features compared with traditional nuclear transformation technology. However, only a handful of species are able to be successfully transformed. Here, we report an efficient and stable plastid transformation protocol for poplar, an economically important tree species grown worldwide. We transformed the Bacillus thuringiensis cry1C gene into the poplar plastid genome, and homoplasmic transplastomic poplar was obtained after two to three rounds of regeneration under antibiotic selection for 7–12 months. The transplastomic poplar expressing Cry1C insecticidal protein showed the highest accumulation level in young leaves, which reached up to 20.7 μg g-1 fresh weight, and comparatively low levels in mature and old leaves, and hardly detectable levels in non-green tissues, such as phloem, xylem and roots. Transplastomic poplar showed high toxicity to Hyphantria cunea and Lymantria dispar, two notorious forest pests worldwide, without affecting plant growth. These results are the first successful examples of insect-resistant poplar generation by plastid genome engineering and provide a new avenue for future genetic improvement of poplar plants.

Plastid genome engineering is an effective method to generate drought-resistant potato plants accumulating glycine betaine in plastids. Glycine betaine (GB) plays an important role under abiotic stress, and its accumulation in chloroplasts is more effective on stress tolerance than that in cytosol of transgenic plants. Here, we report that the codA gene from Arthrobacter globiformis, which encoded choline oxidase to catalyze the conversion of choline to GB, was successfully introduced into potato (Solanum tuberosum) plastid genome by plastid genetic engineering. Two independent plastid-transformed lines were isolated and confirmed as homoplasmic via Southern-blot analysis, in which the mRNA level of codA was much higher in leaves than in tubers. GB accumulated in similar levels in both leaves and tubers of codA-transplastomic potato plants (referred to as PC plants). The GB content was moderately increased in PC plants, and compartmentation of GB in plastids conferred considerably higher tolerance to drought stress compared to wild-type (WT) plants. Higher levels of relative water content and chlorophyll content under drought stress were detected in the leaves of PC plants compared to WT plants. Moreover, PC plants presented a significantly higher photosynthetic performance as well as antioxidant enzyme activities during drought stress. These results suggested that biosynthesis of GB by chloroplast engineering was an effective method to increase drought tolerance.

Objective To determine if early supplemental intra-articular α2-macroglobulin (A2M) has a chondroprotective effect in a collagen II-induced arthritis (CIA) mice model. Methods DBA/1 mice were randomized into four groups (n = 15/group): (a) CIA + 1.2 μg of A2M; (b) CIA + 0.8 μg of A2M; (c) CIA + 0.4 μg of A2M; (d) vehicle + phosphate-buffered saline (PBS). A2M was injected into right ankles and PBS was injected into the left ankles simultaneously as internal control at days 36, 43 and 50. The CIA inflammation clinical score and ankle thickness were recorded every other day starting on day 21 until sacrifice. Changes in inflammation were monitored by in vivo fluorescence molecular tomography (FMT). Inflammation, cartilage and bone damage were assessed with X-ray, histology and immunohistochemistry. Cartilage and inflammation-related gene expression was quantified by real-time polymerase chain reaction (PCR). Results All mice showed ankle inflammation on day 33. After day 43, lower clinical scores, ankle thickness and Sharp/van der Heijde method scores in A2M-treated ankles compared with PBS-treated ankles. FMT data indicated that the inflammation markers MMPSense and ProSense were significantly elevated in the PBS-treated ankles than A2M-treated ankles. Histology and X-ray analyses indicated that A2M administration resulted in lower levels of inflammatory infiltration and synovial hyperplasia, as well as more typical cartilage and bone organization with increased COL II and Aggrecan staining when compared with PBS-treated ankles. In addition, real-time PCR showed that,matrix metalloproteinase-3, -9, -13, COL X and Runx2 were significantly less expressed in A2M-treated groups than PBS-treated animals. Conclusion Early supplemental intra-articular A2M exerts an anti-inflammatory effect and attenuates cartilage and bone damage in a CIA model.

The endochondral ossification plays a critical role in vertebrate limb development and skeletal homeostasis, where limb mesenchyme cells form an intermediate cartilage scaffold that develops into growth plates and then replaced by bone. Although Indian hedgehog (Ihh) is known to control the hypertrophic differentiation process of chondrocytes, its role from the mesenchyme cells to the early stages of chondrogenesis is unclear. To define the function of Ihh in the mesenchymal cell's early stages of chondrogenesis, we specifically delete Ihh in Prx1-expressed mesenchyme cells at E9.5 using Prx1-Cre;Ihh(fl/fl);Rosa26(-ZsGreen1) mice. We found that deleting Ihh in the mesenchyme cells results in an early and quick ossification of the intermediate cartilage scaffold, causing the growth plate and phalange joint absence, short limbs, and dwarfishness. The green fluorescent protein (GFP)-positive cells derived from deleted Ihh mesenchyme cells overlap with von Kossa- and osteocalcin-positive staining area. These deleted Ihh/GFP-positive cells isolated from Prx1-Cre;Ihh(fl/fl);Rosa26(-ZsGreen1) newborn mice had osteogenic differentiation by showing a positive Alizarin red and von Kossa staining, as well as an enhanced Col1a1, osteocalcin, and Runx2 expression. Our findings demonstrate that deleting Ihh in mesenchyme cells during early limb development promotes intermediate cartilage scaffold ossification, which prevents growth plate formation that causes phalange joint absence, short limb, and dwarfish phenotype.

Aim DNA barcoding has been widely applied to species diversity assessment in various ecosystems, including temperate forests, subtropical forests, and tropical rain forests. However, tropical coral islands have never been barcoded before due to the difficulties in field exploring. This study aims at barcoding the flowering plants from a unique ecosystem of the tropical coral islands in the Pacific Ocean and supplying valuable evolutionary information for better understanding plant community assembly of those particular islands in the future. Location Xisha Islands, China. Methods This study built a DNA barcode database for 155 plant species from the Xisha Islands using three DNA markers (ITS, rbcL, and matK). We applied the sequence similarity method and a phylogenetic-based method to assess the barcoding resolution. Results All the three DNA barcodes showed high levels of PCR success (96%-99%) and sequencing success (98%-100%). ITS performed the highest rate of species resolution (>95%) among the three markers, while plastid markers delivered a relatively poor species resolution (85%-90%). Our analyses obtained a marginal increase in species resolution when combining the three DNA barcodes. Main conclusions This study provides the first plant DNA barcode data for the unique ecosystem of tropical coral islands and considerably supplements the DNA barcode library for the flowering plants on the oceanic islands. Based on the PCR and sequencing success rates, and the discriminatory power of the three DNA regions, we recommend ITS as the most successful DNA barcode to identify the flowering plants from Xisha Islands. Due to its high sequence variation and low fungal contamination, ITS could be a preferable candidate of DNA barcode for plants from other tropical coral islands as well. Our results also shed lights on the importance of biodiversity conservation of tropical coral islands.

Fungi have a huge biodiversity and play important roles in soil biogeochemical cycling in island ecosystems. Although island biogeography has been widely studied for macroorganisms, fungal community assembly in true islands and its relationship with island area are less documented. We examined soil fungal communities in 18 oceanic islands of two types (eight non-coral islands and 10 coral islands) using the Illumina MiSeq sequencing technique. Our results showed that fungal α-diversity (species richness) was substantially different among the oceanic islands, with a higher value in non-coral islands than in coral islands. Fungal α-diversity was significantly affected by soil potassium and magnesium (Mg) and plant communities in non-coral islands, whereas only soil Mg significantly affected it in coral islands. Soil fungal community composition was significantly different in the non-coral and coral islands and was influenced by soil property, plant community and spatial distance. The ecological stochasticity model showed that the fungal community assembly was mainly governed by deterministic processes regardless of island type. Fungal β-diversity, but not α-diversity, increased significantly with increasing island area. These findings have implications for the better prediction of soil fungal community dynamics in island systems and biodiversity conservation in fragmented habitats.

Photorespiratory hydrogen peroxide (H2 O2 ) plays key roles in pathogenesis responses by triggering the salicylic acid (SA) pathway in Arabidopsis. However, factors linking intracellular H2 O2 to activation of the SA pathway remain elusive. In this work, the catalase-deficient Arabidopsis mutant, cat2, was exploited to elucidate the impact of S-nitrosoglutathione reductase 1 (GSNOR1) on H2 O2 -dependent signalling pathways. Introducing the gsnor1-3 mutation into the cat2 background increased S-nitrosothiol levels and abolished cat2-triggered cell death, SA accumulation, and associated gene expression but had little additional effect on the major components of the ascorbate-glutathione system or glycolate oxidase activities. Differential transcriptome profiles between gsnor1-3 and cat2 gsnor1-3 together with damped ROS-triggered gene expression in cat2 gsnor1-3 further indicated that GSNOR1 acts to mediate the SA pathway downstream of H2 O2 . Up-regulation of GSNOR activity was compromised in cat2 cad2 and cat2 pad2 mutants in which glutathione accumulation was genetically prevented. Experiments with purified recombinant GSNOR revealed that the enzyme is posttranslationally regulated by direct denitrosation in a glutathione-dependent manner. Together, our findings identify GSNOR1-controlled nitrosation as a key factor in activation of the SA pathway by H2 O2 and reveal that glutathione is required to maintain this biological function.

Owing to their sessile life habit, plants are continuously subjected to a broad range of environmental stresses. During periods of (a)biotic stresses, reactive oxygen species (ROS) levels can rise excessively, leading to oxidative stress. Glutathione reductase (GR) plays an important role in scavenging the ROS and maintenance of redox potential of the cell during oxidative stress. To enhance ROS scavenging capacity, and hence stress tolerance, the Arabidopsis thalianaGR2 (AtGR2) gene was expressed from the tobacco plastid (chloroplast) genome, the main source of ROS production in plant photosynthetic tissues, in this study. Leaves of transplastomic tobacco plants had about seven times GR activity and 1.5 times total glutathione levels compared to wild type. These transplastomic tobacco plants showed no discernible phenotype and exhibited more tolerance to methyl viologen-induced oxidative stress than wild-type control plants. The results indicate that introducing AtGR2 in chloroplasts is an efficient approach to increase stress tolerance. This study also provides evidence that increasing antioxidant enzyme via plastid genome engineering is an alternative to enhance plant’s tolerance to stressful conditions.

The Bacillus thuringiensis (Bt) cry3Bb gene was successfully introduced into poplar plastid genome, leading to transplastomic poplar with high mortality to Plagiodera versicolora. Poplar (Populus L.) is one of the main resource of woody industry, but being damaged by insect pests. The feasibility and efficiency of plastid transformation technology for controlling two lepidopteran caterpillars have been demonstrated previously. Here, we introduced B. thuringiensis (Bt) cry3Bb into poplar plastid genome by biolistic bombardment for controlling P. versicolora, a widely distributed forest pest. Chimeric cry3Bb gene is controlled by the tobacco plastid rRNA operon promoter combined with the 5′UTR from gene10 of bacteriophage T7 (NtPrrn:T7g10) and the 3′UTR from the E. coli ribosomal RNA operon rrnB (TrrnB). The integration of transgene and homoplasmy of transplastomic poplar plants was confirmed by Southern blot analysis. Northern blot analysis indicated that cry3Bb was transcribed to both read through and shorter length transcripts in plastid. The transplastomic poplar expressing Cry3Bb insecticidal protein showed the highest accumulation level in young leaves, which reach up to 16.8 μg/g fresh weight, and comparatively low levels in mature and old leaves. Feeding the young leaves from Bt-Cry3Bb plastid lines to P. versicolora caused 100% mortality in the first-instar larvae after only 1 day, in the second-instar larvae after 2 days, and in the third-instar larvae for 3 days. Thus, we report a successful extension of plastid engineering poplar against the chrysomelid beetle.

The present paper deals with the detailed cytological study of Suriana maritima L. (Surianaceae) from the Xisha Islands (also known as Paracel Islands) of South China Sea. The somatic chromosome nu...

Under natural conditions, plants constantly encounter various fluctuating environmental stresses, which potentially restrict plant growth, plant development and even limit crop productivity. In addition to carbon fixation activity in C4 photosynthesis, NADP-dependent malic enzyme (NADP-ME) has been suggested to play important roles in diverse stress responses in plants. NADP-ME is one of the essential enzymes metabolizing malate, which is important for stabilizing cytoplasmic pH, controlling stomatal aperture, increasing resistance to aluminum excess and pathogen. Pyruvate, another product of NADP-ME reaction, participates in the synthesis of defense compounds such as flavonoids and lignin, which are involved in stresses tolerance such as mechanical wounding and pathogen invasion. Moreover, NADP-ME provides essential reductive coenzyme NADPH in the biosynthesis of flavonoids and lignin. On the other hand, NADPH is crucial for reactive active species (ROS) metabolizing systems such as the ascorbate-glutathione pathway and NADPH-dependent thioredoxin reductase, and is also required by apoplastic oxidative burst in most plant-pathogen interactions. This mini-review is largely focus on the characteristics of gene expression and activity of NADP-ME, as well as its interaction with ROS signaling under a variety of biotic and abiotic stress responses, which will provide a theoretical foundation for breeding of stress resistant crops.

RNA interference (RNAi) has emerged as a novel and feasible strategy for pest management. Methods for cost-effective production and stable delivery of double-stranded RNA (dsRNA) to the target insects are crucial for the wide application of RNAi for pest control. In this study, we tested the expression of dsRNA in RNaseIII-deficient Escherichia coli HT115 which was then fed to Plagiodera versicolora larvae, an insect pest of Salicaceae plants worldwide. By targeting six potential genes, including actin (ACT), signal recognition particle protein 54k (SRP54), heat shock protein 70 (HSC70), shibire (SHI), cactus (CACT), and soluble N-ethylmaleimide-sensitive fusion attachment proteins (SNAP), we found that feeding bacteria-expressed dsRNA successfully triggered the silencing of the five target genes tested and the suppression of ACT and SRP54 genes caused significant mortality. Our results suggest that the oral delivery of bacteria-expressed dsRNA is a potential alternative for the control of P. versicolora, and that ACT and SRP54 genes are the potent targets.

The objective of this study was to determine if Ihh is required for fracture healing. Fibular fracture was created in adult Col2a1-CreERT2; Ihhfl/fl mice. Ihhfl/fl mice received Tamoxifen (TM) to delete Ihh. WT mice received Cyclopamine to inhibit Hh pathway. Callus tissue properties and Ihh pathway were analyzed at 1, 2, and 3 weeks post-fracture by X-ray, micro-CT, mechanical test, RT-PCR and immunohistochemistry. Deleted Ihh was evidenced by the occurrence of growth plate closure in the Ihhfl/fl mice by X-ray 3 weeks after TM treatment. All mice showed fracture healing at 3 weeks post-operation. Histology analysis indicated that, compared to the control, cartilage area was less in fracture sites from Ihh deficient animals by either genetic deletion or drug inhibition at 1 and 2 weeks post-fracture. Ihh immunostaining and its mRNA level were diminished in the fracture callus in Ihh reduced mice. There was no significant difference in BV/TV, BMD and mechanical test. Interruption to Ihh pathway by either genetic or pharmaceutical approach didn’t affect fibular fracture healing in these mice. This surprised finding implicates that the deleted Ihh does not affect fracture healing in this model.

Plastid transformation for the expression of recombinant proteins and entire metabolic pathways has become a promising tool for plant biotechnology. However, large-scale application of this technology has been hindered by some technical bottlenecks, including lack of routine transformation protocols for agronomically important crop plants like rice or maize. Currently, there are no standard or commercial plastid transformation vectors available for the scientific community. Construction of a plastid transformation vector usually requires tedious and time-consuming cloning steps. In this study, we describe the adoption of an in vivo Escherichia coli cloning (iVEC) technology to quickly assemble a plastid transformation vector. The method enables simple and seamless build-up of a complete plastid transformation vector from five DNA fragments in a single step. The vector assembled for demonstration purposes contains an enhanced green fluorescent protein (GFP) expression cassette, in which the gfp transgene is driven by the tobacco plastid ribosomal RNA operon promoter fused to the 5’ untranslated region (UTR) from gene10 of bacteriophage T7 and the transcript-stabilizing 3’UTR from the E. coli ribosomal RNA operon rrnB. Successful transformation of the tobacco plastid genome was verified by Southern blot analysis and seed assays. High-level expression of the GFP reporter in the transplastomic plants was visualized by confocal microscopy and Coomassie staining, and GFP accumulation was approximately 9% of the total soluble protein. The iVEC method represents a simple and efficient approach for construction of plastid transformation vector, and offers great potential for the assembly of increasingly complex vectors for synthetic biology applications in plastids.

The complexity of plant antioxidative systems gives rise to many unresolved questions. One relates to the functional importance of dehydroascorbate reductases (DHARs) in interactions between ascorbate and glutathione. To investigate this issue, we produced a complete set of loss-of-function mutants for the three annotated Arabidopsis (Arabidopsis thaliana) DHARs. The combined loss of DHAR1 and DHAR3 expression decreased extractable activity to very low levels but had little effect on phenotype or ascorbate and glutathione pools in standard conditions. An analysis of the subcellular localization of the DHARs in Arabidopsis lines stably transformed with GFP fusion proteins revealed that DHAR1 and DHAR2 are cytosolic while DHAR3 is chloroplastic, with no evidence for peroxisomal or mitochondrial localizations. When the mutations were introduced into an oxidative stress genetic background (cat2), the dhar1 dhar2 combination decreased glutathione oxidation and inhibited cat2-triggered induction of the salicylic acid pathway. These effects were reversed in cat2 dhar1 dhar2 dhar3 complemented with any of the three DHARs. The data suggest that (1) DHAR can be decreased to negligible levels without marked effects on ascorbate pools, (2) the cytosolic isoforms are particularly important in coupling intracellular hydrogen peroxide metabolism to glutathione oxidation, and (3) DHAR-dependent glutathione oxidation influences redox-driven salicylic acid accumulation.

Glutathione is a pivotal molecule in oxidative stress, during which it is potentially oxidized by several pathways linked to H2O2 detoxification. We have investigated the response and functional importance of 3 potential routes for glutathione oxidation pathways mediated by glutathione S-transferases (GST), glutaredoxin-dependent peroxiredoxins (PRXII), and dehydroascorbate reductases (DHAR) in Arabidopsis during oxidative stress. Loss-of-function gstU8, gstU24, gstF8, prxIIE and prxIIF mutants as well as double gstU8 gstU24, gstU8 gstF8, gstU24 gstF8, prxIIE prxIIF mutants were obtained. No mutant lines showed marked changes in their phenotype and glutathione profiles in comparison to the wild-type plants in either optimal conditions or oxidative stress triggered by catalase inhibition. By contrast, multiple loss of DHAR functions markedly decreased glutathione oxidation triggered by catalase deficiency. To assess whether this effect was mediated directly by loss of DHAR enzyme activity, or more indirectly by upregulation of other enzymes involved in glutathione and ascorbate recycling, we measured expression of glutathione reductase (GR) and expression and activity of monodehydroascorbate reductases (MDHAR). No evidence was obtained that either GRs or MDHARs were upregulated in plants lacking DHAR function. Hence, interplay between different DHARs appears to be necessary to couple ascorbate and glutathione pools and to allow glutathione-related signaling during enhanced H2O2 metabolism.

NADPH is a pivotal molecule in oxidative stress, during which it is potentially produced by several cytosolic NADP-linked dehydrogenases. This study investigated the response and functional importance of the major leaf cytosolic NADP-malic enzyme in Arabidopsis (NADP-ME2) during oxidative stress. Data from both microarray and targeted quantitative PCR analyses showed that NADP-ME2 transcripts accumulated in response to ozone or in mutants undergoing intracellular oxidative stress. To test the functional importance of this response, loss-of-function nadp-me2 mutants were obtained and the effects of oxidative stress of intracellular and extracellular origin were tested. Despite much decreased leaf NADP-ME activity, nadp-me2 showed a wild-type phenotype when exposed to ozone. Introduction of the nadp-me2 mutations into the catalase-deficient cat2 background did not alter growth inhibition or lesions triggered by intracellular oxidative stress. Similarly, loss of NADP-ME2 function had little effect on cat2-triggered changes in glutathione or NADPH. While single nadp-me2 mutations produced slight effects on basal resistance to one type of bacteria, they did not affect resistance induced by the cat2 mutation. Taken together, the results suggest that, although NADP-ME2 induction is part of the response to oxidative stress, the enzyme is not an essential determinant of the outcome of such stress.