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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.

Nicotinamide adenine dinucleotide (NAD) is a pivotal coenzyme that has emerged as a central hub linking redox equilibrium and signal transduction in living cells. The homeostasis of NAD is required for plant growth, development, and adaption to environmental stresses. Quinolinate phosphoribosyltransferase (QPRT) is a key enzyme in NAD de novo synthesis pathway. T-DNA-based disruption of QPRT gene is embryo lethal in Arabidopsis thaliana. Therefore, to investigate the function of QPRT in Arabidopsis, we generated transgenic plants with decreased QPRT using the RNA interference approach. While interference of QPRT gene led to an impairment of NAD biosynthesis, the QPRT RNAi plants did not display distinguishable phenotypes under the optimal condition in comparison with wild-type plants. Intriguingly, they exhibited enhanced sensitivity to an avirulent strain of Pseudomonas syringae pv. tomato (Pst-avrRpt2), which was accompanied by a reduction in salicylic acid (SA) accumulation and down-regulation of pathogenesis-related genes expression as compared with the wild type. Moreover, oxidative stress marker genes including GSTU24, OXI1, AOX1 and FER1 were markedly repressed in the QPRT RNAi plants. Taken together, these data emphasized the importance of QPRT in NAD biosynthesis and immunity defense, suggesting that decreased antibacterial immunity through the alteration of NAD status could be attributed to SA- and reactive oxygen species-dependent pathways.

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.

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.

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.

International audience; Plants optimize their growth and survival through highly integrated regulatory networks that coordinate defensive measures and developmental transitions in response to environmental cues. Protein phosphatase 2A (PP2A) is a key signaling component that controls stress reactions and growth at different stages of plant development, and the PP2A regulatory subunit PP2A-B'gamma is required for negative regulation of pathogenesis responses and for maintenance of cell homeostasis in short-day conditions. Here, we report molecular mechanisms by which PP2A-B'gamma regulates Botrytis cinerea resistance and leaf senescence in Arabidopsis (Arabidopsis thaliana). We extend the molecular functionality of PP2A-B'gamma to a protein kinase-phosphatase interaction with the defense-associated calcium-dependent protein kinase CPK1 and present indications this interaction may function to control CPK1 activity. In presenescent leaf tissues, PP2A-B'gamma is also required to negatively control the expression of salicylic acid-related defense genes, which have recently proven vital in plant resistance to necrotrophic fungal pathogens. In addition, we find the premature leaf yellowing of pp2a-b'gamma depends on salicylic acid biosynthesis via SALICYLIC ACID INDUCTION DEFICIENT2 and bears the hallmarks of developmental leaf senescence. We propose PP2A-B'gamma age-dependently controls salicylic acid-related signaling in plant immunity and developmental leaf senescence.

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...

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.

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.

Summary Oxidative stress responses are influenced by growth day length, but little is known about how this occurs. A combined reverse genetics, metabolomics and proteomics approach was used to address this question in Arabidopsis thaliana. A catalase-deficient mutant (cat2), in which intracellular oxidative stress drives pathogenesis-related responses in a day length-dependent manner, was crossed with a knockdown mutant for a specific type 2A protein phosphatase subunit (pp2a-b′γ). In long days (LD), the pp2a-b′γ mutation reinforced cat2-triggered pathogenesis responses. In short days (SD), conditions in which pathogenesis-related responses were not activated in cat2, the additional presence of the pp2a-b′γ mutation allowed lesion formation, PATHOGENESIS-RELATED GENE1 (PR1) induction, salicylic acid (SA) and phytoalexin accumulation and the establishment of metabolite profiles that were otherwise observed in cat2 only in LD. Lesion formation in cat2 pp2a-b′γ in SD was genetically dependent on SA synthesis, and was associated with decreased PHYTOCHROME A transcripts. Phosphoproteomic analyses revealed that several potential protein targets accumulated in the double mutant, including recognized players in pathogenesis and key enzymes of primary metabolism. We conclude that the cat2 and pp2a-b′γ mutations interact synergistically, and that PP2A-B′γ is an important player in controlling day length-dependent responses to intracellular oxidative stress, possibly through phytochrome-linked pathways.

A new species of Quercus (Fagaceae), Q. pseudosetulosa is described and illustrated from Dawanshan Island in Guangdong, China. The new species belongs to the Q. setulosa morpho-complex with glabrous leaf abaxial surface and acuminate tip, but it differs from other related species by its leaf with long petioles, large, thickly leathery, glabrous, the irregular spaced secondary veins, the inconspicuous tertiary veins and the recurved leaf blades. Its distribution, habitats and conservation status are also addressed.

To establish a successful in vitro plant regeneration system in Alternanthera philoxeroides (Mart.) Griseb, an orthogonal design was used to investigate the effects of three factors (plant growth regulators, explant types and dark treatment in initial-stage), each having three levels. The effects of these factors and levels on callus induction and shoot regeneration were quantitatively evaluated by analysis of variance. The experimental results showed that the callus induction was significantly affected by 2, 4-dichlorophenoxyacetic acid (2, 4-D), and shoot differentiation from subcultured pieces of callus was enhanced mostly by dark treatment in initial-stage. The optimal conditions for callus induction are obtained from the stem explants cultured on semi-solid Murashige and Skoog (MS) medium plus 2.2 μM BA and 2.2 μM 2, 4-D, with 20 days dark treatment in initial-stage. The highest frequency of shoot regeneration is obtained from the calli cultured on semi-solid MS medium plus 8.8 μM BA, without dark treatment in initial-stage.

Stillingia (Euphorbiaceae) contains ca. 30 species from Latin America, the southern United States, and various islands in the tropical Pacific and in the Indian Ocean. We report here for the first time the occurrence of a member of the genus in China, Stillingia lineata subsp. pacifica. The distribution of the genus in China is apparently narrow, known only from Pingzhou and Wanzhou Islands of the Wanshan Archipelago in the South China Sea, which is close to the Pearl River estuary. This study updates our knowledge on the geographic distribution of the genus, and provides new palynological data as well.

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.

NAD and NADP are key brokers in cell redox and energy metabolism, but they also play important roles in signalling pathways. Several of these latter roles involve NAD cleavage, requiring continuous resynthesis of the molecule to maintain cellular pools. Mammals synthesize their cellular NAD from pyridine precursors that are obtained as niacin (vitamin B 3 ) or produced from tryptophan. In many foods, the major source of niacin can be the pyridine ring of NAD and NADP. Plant NAD contents show considerable plasticity in response to environmental conditions, and recent advances have opened up new perspectives for rational manipulation of tissue NAD contents. In the model plant Arabidopsis, the de novo pathway of plant NAD synthesis has been identified, as have proteins involved in the subcellular transport of NAD and in NAD cleavage and recycling. Here, we discuss emerging concepts on the regulation of NAD contents in plants, and we report the use of transformation technology to enrich NAD several-fold in plant tissues through overexpression of the de novo synthesis pathway. Microarray analyses reveal that these increases trigger a characteristic transcriptomic signature, notably involving specific genes in defence phytohormone signalling. Thus, manipulation of plant NAD biosynthesis may offer interesting perspectives to (1) dissect redox-related signalling in stress responses, (2) enhance the nutritional value of plants, and (3) modify plant resistance to environmental stress.

Immature embryos have been used frequently as target tissues in the genetical transformation of wheat. However, obtaining a large number of high quality immature embryos throughout the year is a laborious and delicate process, because of the need to cultivate the plants under controlled conditions. To circumvent this, we have employed mature embryos rather than immature ones as starter explants for Agrobacterium-mediated transformation of an elite wheat (Triticum aestivum L.) cultivar EM12. The neomycin phosphotransferase II, (npt II) and beta-glucuronidase (gus) genes were used as selectable and screenable marker genes, respectively, to assess and optimize the performance of T-DNA delivery. With the aid of an orthogonal design, the effect of four factors in combination on transfer DNA (T-DNA) delivery was studied. These factors were preculture duration, different kinds of inoculation, length of inoculation and co-culture condition. Optimal conditions for T-DNA delivery were obtained for mature embryos precultured for 14 days, followed by immersing in inoculation suspension with full strength Murashige and Skoog (MS) salts in darkness at 23-25 degrees C for 3 h, and then co-culturing with Agrobacterium under desiccating condition in the dark at 23-24 degrees C for 2-3 days. Complete analysis of transgene insertion demonstrated that the optimized method for Agrobacterium-mediated transformation of mature embryos of wheat was efficient and practicable.