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12-oxo-Phytodienoic acid (OPDA) is a primary precursor of (-)-jasmonic acid (JA), able to trigger autonomous signaling pathways that regulate a unique subset of jasmonate-responsive genes, activating and fine-tuning defense responses, as well as growth processes in plants. Recently, a number of studies have illuminated the physiol-molecular activities of OPDA signaling in plants, which interconnect the regulatory loop of photosynthesis, cellular redox homeostasis, and transcriptional regulatory networks, together shedding new light on (i) the underlying modes of cellular interfaces between growth and defense responses (e.g., fitness trade-offs or balances) and (ii) vital information in genetic engineering or molecular breeding approaches to upgrade own survival capacities of plants. However, our current knowledge regarding its mode of actions is still far from complete. This review will briefly revisit recent progresses on the roles and mechanisms of OPDA and information gaps within, which help in understanding the phenotypic and environmental plasticity of plants.

Plant-parasitic nematodes (PPN) are among the most economically and ecologically damaging pests, causing severe losses of crop production worldwide. Chemical-based nematicides have been widely used, but these may have adverse effects on human health and the environment. Hence, biological control agents (BCAs) have become an alternative option for controlling PPN, since they are environmentally friendly and cost effective. Lately, a major effort has been made to evaluate the potential of a commercial grade strain of plant growth-promoting rhizobacteria (PGPR) as BCAs, because emerging evidence has shown that PGPR can reduce PPN in infested plants through direct and/or indirect antagonistic mechanisms. Direct antagonism occurs by predation, release of antinematicidal metabolites and semiochemicals, competition for nutrients, and niche exclusion. However, the results of direct antagonism may be inconsistent due to unknown endogenous and exogenous factors that may prevent PGPR from colonizing plant’s roots. On the other hand, indirect antagonism may occur from the induced systemic resistance (ISR) that primes whole plants to better fight against various biotic and abiotic constraints, actuating faster and/or stronger defense responses (adaption), enhancing their promise as BCAs. Hence, this review will briefly revisit (i) two modes of PGPR in managing PPN, and (ii) the current working models and many benefits of ISR, in the aim of reassessing current progresses and future directions for isolating more effective BCAs and/or developing better PPN management strategy.

In recent decades, drought has become a global problem for food security and agricultural production. A variety of strategies have been developed to enhance drought tolerance, but largely unsuccessful since most drought-responsive genes (DRGs) stimulate a stomata closure and in turn suppress plant growth and yield. To access if and/or how plants could enhance drought tolerance without trading off growth and development, we screened and isolated a plant growth-promoting rhizobacterium, Paenibacillus polymyxa CR1, capable of 1) priming drought tolerance and concurrently 2) increasing root growth in plants, e.g., Arabidopsis and soybean. In parallel, we uncovered that P. polymyxa CR1 3) induces the expression of two DRGs, Response to Desiccation (RD)29A and RD29B, 4) of which pattern upregulations are controlled by a diurnal rhythm. Besides, RD29A and RD29B act as 5) 'memory' genes; their transcript levels are increased to a greater extent when plants encountered P. polymyxa CR1 for the second time compared to an initial exposure. In line with these findings, T-DNA insertion mutant Arabidopsis of RD29A or RD29B displayed enhanced susceptibility to drought, without any change in stomata behaviors or growth rates, than wild-type plants. Hence, we conclude that RD29A or RD29B are unique, efficacious generic materials that can potentially aid in upgrading the plants own survival capacity against drought without reducing yield potential.

Plant parasitic nematodes (PPN) are microscopic roundworms that mostly develop an obligate parasitic relationship with hosts. Once PPN reach root surfaces, they slowly insert a stylet, needle-like structure, into and feed cytosolic nutrients from roots, which cause cell and tissue mortality (Fosu-Nyarko and Jones, 2016; Mitchum et al., 2013). Recently, these PPN-associated damages have become of great agricultural and economic importance resulting in an estimated annual loss of ∼14 percent of world crop production (Nicol et al., 2011). Thus, an urgent breakthrough is needed in developing effective and sustainable pest management programs such as new resistance cultivars. It is however not necessarily forthcoming, largely due to our little knowledge of the defense physiology of plants against PPN. Hence, this review will briefly revisit current working models of plant and PPN interactions with a focus of plant defense responses, and pinpoint information gaps within, which help revamp unique and alternative prospective for the future studies. Keywords: Plant parasitic nematodes (PPN), Hypersensitive response, Host specificity of PPN, Chemotaxis, Root exudates

SUMMARY Abscisic acid (ABA) is an important phytohormone controlling responses to abiotic stresses and is sensed by proteins from the PYR/PYL/RCAR family. To explore the genetic contribution of PYLs toward ABA-dependent and ABA-independent processes, we generated and characterized high-order Arabidopsis mutants with mutations in the PYL family. We obtained a pyl quattuordecuple mutant and found that it was severely impaired in growth and failed to produce seeds. Thus, we carried out a detailed characterization of a pyl duodecuple mutant, pyr1pyl1/2/3/4/5/7/8/9/10/11/12. The duo-decuple mutant was extremely insensitive to ABA effects on seed germination, seedling growth, stomatal closure, leaf senescence, and gene expression. The activation of SnRK2 protein kinases by ABA was blocked in the duodecuple mutant, but, unexpectedly, osmotic stress activation of SnRK2s was enhanced. Our results demonstrate an important role of basal ABA signaling in growth, senescence, and abscission and reveal that PYLs antagonize ABA-independent activation of SnRK2s by osmotic stress., In Brief Zhao et al. generated duodecuple and quattuordecuple Arabidopsis PYL ABA receptor mutants. Characterization of the mutants revealed that the ABA receptors are critical for plant growth and development and negatively regulate ABA-independent SnRK2 activity by interacting with and inhibiting osmotic stress-activated SnRK2 protein kinases.

Plant parasitic nematodes (PPN) are microscopic soil herbivores that cause damage to many economic crops. For the last century, it has been proposed that chemotaxis is the primary means by which PPN locate host plant roots. The identities and modes of action of chemoattractants that deliver host-specific messages to PPN, however, are still elusive. In this study, a unique multidimensional agar-based motility assay was developed to assess the impacts of root exudates on the short-range motility and orientation of PPN. Three PPN (Rotylenchulus reniformis, Meloidogyne incognita and Heterodera glycines) and root exudates from their respective host and non-host plants (cotton, soybean, and peanut) were used to validate the assay. As predicted, R. reniformis and M. incognita were attracted to root exudates of cotton and soybean (hosts), but not to the exudates of peanut (non-host). Likewise, H. glycines was attracted to soybean (host) root exudates. These results underpinned the intrinsic roles of root exudates in conveying the host specificity of PPN. In particular, PPN selectively identified and targeted to hydrophilic, but not hydrophobic, fractions of root exudates, indicating that groundwater should be an effective matrix for chemotaxis associated with PPN and their host plant interactions.

Posttranslational modification by a reduced glutathione determines distinctive quaternary structures and functions between two 2-cysteine peroxiredoxin A and B in the chloroplasts., In plants, growth-defense trade-offs occur because of limited resources, which demand prioritization towards either of them depending on various external and internal factors. However, very little is known about molecular mechanisms underlying their occurrence. Here, we describe that cyclophilin 20-3 (CYP20-3), a 12-oxo-phytodienoic acid (OPDA)–binding protein, crisscrosses stress responses with light-dependent electron reactions, which fine-tunes activities of key enzymes in plastid sulfur assimilations and photosynthesis. Under stressed states, OPDA, accumulates in the chloroplasts, binds and stimulates CYP20-3 to convey electrons towards serine acetyltransferase 1 (SAT1) and 2-Cys peroxiredoxin A (2CPA). The latter is a thiol-based peroxidase, protecting and optimizing photosynthesis by reducing its toxic byproducts (e.g., H2O2). Reduction of 2CPA then inactivates its peroxidase activity, suppressing the peroxide detoxification machinery, whereas the activation of SAT1 promotes thiol synthesis and builds up reduction capacity, which in turn triggers the retrograde regulation of defense gene expressions against abiotic stress. Thus, we conclude that CYP20-3 is a unique metabolic hub conveying resource allocations between plant growth and defense responses (trade-offs), ultimately balancing optimal growth phonotype.

DNA methylation confers epigenetic regulation on gene expression and thereby on various biological processes. Tomato has emerged as an excellent system to study the function of DNA methylation in plant development. To date, regulation and function of DNA methylation maintenance remains unclear in tomato plants. Here, we report the critical function of tomato (Solanum lycopersicum) Methyltransferase 1 (SlMET1) in plant development and DNA methylome and transcriptome regulation. Using CRISPR-Cas9 gene editing, we generated slmet1 mutants and observed severe developmental defects with a frame-shift mutation, including small and curly leaves, defective inflorescence, and parthenocarpy. In leaf tissues, mutations in SlMET1 caused CG hypomethylation and CHH hypermethylation on a whole-genome scale, leading to a disturbed transcriptome including ectopic expression of many RIN target genes such as ACC2 in leaf tissues, which are normally expressed in fruits. Neither the CG hypomethylation nor CHH hypermethylation in the slmet1 mutants is related to tissue culture. Meanwhile, tissue culture induces non-CG hypomethylation, which occurs more frequently at gene regions than at TE regions. Our results depict SlMET1- and tissue culture-dependent tomato DNA methylomes, and that SlMET1 is required for maintaining a normal transcriptome and normal development of tomato.

Mitogen-activated protein kinase cascades are highly conserved signaling modules downstream of receptors/sensors and play pivotal roles in signaling plant defense against pathogen attack. Extensive studies on Arabidopsis MPK4 have implicated that the MAP kinase is involved in multilayered plant defense pathways. In this study, we identified tobacco NtMPK2 as an ortholog of AtMPK4. Transgenic tobacco overexpressing NtMPK2 markedly enhances resistance to Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) virulent and avirulent strains. Transcriptome analysis of NtMPK2-dependent genes shows that possibly the basal resistance system is activated by NtMPK2 overexpression. In addition to NtMPK2-mediated resistance, multiple pathways are involved in response to the avirulent bacteria based on analysis of Pst-responding genes, including SA and ET pathways. Notably, it is possible that biosynthesis of antibacterial compounds is responsible for inhibition of Pst DC3000 avirulent strain when programmed cell death processes in the host. Our results uncover that NtMPK2 positively regulate tobacco defense response to Pst DC3000 and improve our understanding of plant molecular defense mechanism.

The Pacific geoduck Panopea generosa is the largest burrowing clam in the world and adults can live up to a meter below the sediment surface. To extract these clams, harvesters use pressurized water jets to dislodge surrounding sediments. This type of disturbance could have significant effects on the local benthic environment, but has been little examined. The present study was conducted on one intertidal and one subtidal plot to assess potential effects of commercial-scale geoduck harvesting on the sedimentary benthic environment and nearby eelgrass beds. Sediment samples were collected inside the impacted plots and at intervals up to 75 m away while eelgrass sampleswere collected adjacent to the impacted plots and at intervals up to 50maway, seasonally over 2 y.Harvest of the subtidal plot occurred at one year and mock harvest of the intertidal plot occurred after one preimpact sample. Sediment and infaunal qualities examined included: grain size, percent organics, total nitrogen, total organic...

The newly developed CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system has emerged as an efficient tool for genome-editing, providing an alternative to classical mutagenesis and transgenic methods to study gene function and improve crop traits. CRISPR/Cas facilitates targeted gene editing through RNA-guided DNA cleavage followed by cellular DNA repair mechanisms that introduce sequence changes at the site of cleavage. Here we describe a detailed procedure for our previously developed and highly efficient CRISPR/Cas9 method that allows the generation of heritable-targeted gene mutations and corrections in Arabidopsis. This protocol describes the strategies and steps for the selection of targets, design of single-guide RNA (sgRNA), vector construction and analysis of transgenic lines. We also offer a method to target two loci simultaneously using vectors containing two different sgRNAs. The principles described in this protocol can be applied to other plant species to generate stably inherited DNA modifications.

The production of live microalgae can be a critical constraint to economic bivalve culture due to high production cost, culture instability, and batch variability. The integration of alternatives to live algae is consequently a current trend in bivalve aquaculture, but dietary alternatives remain unexplored for the culture of the Pacific geoduck clam ( Panopea generosa ). To investigate the feasibility of substitution of live algae for juvenile geoduck feeding, the present study replaced a positive control microalgal diet [ Chaetoceros muelleri (CM) and Tisochrysis lutea (TISO)] with various levels (0, 25, 50, 75, 100% substitution by ash-free dry weight) of the spray-dried heterotrophic thraustochytrid Schizochytrium sp. or the spray-dried cyanobacterium Spirulina ( Arthrospira platensis ). Feeding trials (14 days) were conducted on two sizes of juvenile geoducks [mean initial shell length: 2.47 mm (trial 1); 4.00 mm (trial 2)]. Geoduck growth and survival were measured to quantify treatment success and proximate (total protein, carbohydrate, lipid) and fatty acid analyses were conducted on diets and final geoduck tissues. Growth rate was significantly higher in individuals fed Spirulina than in those given Schizochytrium sp., but algal-replacement treatments resulted in significantly reduced growth in comparison to the control of live algae (i.e. 0% substitution) at substitution levels of 25% and higher or 50% and higher, depending on geoduck size and growth variable. The superior diet of live microalgae contained a polyunsaturated fatty acid Σn–3 / n–6 ratio of 2.9, a value which appears to be conserved in the bivalve literature (range: 2.0–3.3). Tissue analyses suggested limited digestion of spray-dried derived protein and carbohydrate, but indicated general lipid/fatty acid sequestration. Based on these findings, the dietary incorporation of spray-dried Schizochytrium or Spirulina in juvenile geoduck culture is not recommended. It is proposed that additional potential dietary alternatives attempt to mirror the nutritional composition of the combination of the live microalgae CM and TISO since this diet elicited the best growth rate in both the present study and previous research examining the effect of various microalgal species on juvenile geoduck growth.

CRISPR/Cas9 and TALEN are currently the two systems of choice for genome editing. We have studied the efficiency of the TALEN system in rice as well as the nature and inheritability of TALEN-induced mutations and found important features of this technology. The N287C230 TALEN backbone resulted in low mutation rates (0-6.6%), but truncations in its C-terminal domain dramatically increased efficiency to 25%. In most transgenic T0 plants, TALEN produced a single prevalent mutation accompanied by a variety of low-frequency mutations. For each independent T0 plant, the prevalent mutation was present in most tissues within a single tiller as well as in all tillers examined, suggesting that TALEN-induced mutations occurred very early in the development of the shoot apical meristem. Multigenerational analysis showed that TALEN-induced mutations were stably transmitted to the T1 and T2 populations in a normal Mendelian fashion. In our study, the vast majority of TALEN-induced mutations (~81%) affected multiple bases and ~70% of them were deletions. Our results contrast with published reports for the CRISPR/Cas9 system in rice, in which the predominant mutations affected single bases and deletions accounted for only 3.3% of the overall mutations.

This research examined the individual effects of temperature and ration on growth and survival of juveniles of the Pacific geoduck Panopea generosa Gould, 1850, in two separate experiments. Growth parameters measured included shell length, daily shell increment, individual total body wet weight, specific growth rate, individual total body dry weight, and total body ash-free dry weight (AFDW). Larvae in all treatments were fed a binary microalgal diet of Chaetoceros muelleri and Tisochrysis lutea mixed at a 1:1 ratio by AFDW. The temperature experiment examined the effect of four temperatures (7°C, 11°C, 15°C, and 19°C) using two geoduck cohorts. One cohort was comprised of larger juveniles (mean initial shell length ± SE, 3.22 ± 0.05 mm) and the other included smaller individuals (0.54 ± 0.01 mm) which were cultured for 28 days and 21 days, respectively. Using a separate cohort, the foodration experiment examined the effect of nine rations (0.0, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, 64.0, and 128.0 × 1...

Phytoplasma is an insect-transmitted pathogen that causes witches' broom disease in many plants. Paulownia witches' broom is one of the most destructive diseases threatening Paulownia production. The molecular mechanisms associated with this disease have been investigated by transcriptome sequencing, but changes in protein abundance have not been investigated with isobaric tags for relative and absolute quantitation. Previous results have shown that methyl methane sulfonate (MMS) can help Paulownia seedlings recover from the symptoms of witches' broom and reinstate a healthy morphology. In this study, a transcriptomic-assisted proteomic technique was used to analyze the protein changes in phytoplasma-infected Paulownia tomentosa seedlings, phytoplasma-infected seedlings treated with 20 and 60 mg·L−1 MMS, and healthy seedlings. A total of 2,051 proteins were obtained, 879 of which were found to be differentially abundant in pairwise comparisons between the sample groups. Among the differentially abundant proteins, 43 were related to Paulownia witches' broom disease and many of them were annotated to be involved in photosynthesis, expression of dwarf symptom, energy production, and cell signal pathways.

The jasmonate family of phytohormones plays central roles in plant development and stress acclimation. However, the regulatory modes of their signaling circuitry remain largely unknown. Here we describe that cyclophilin 20–3 (CYP20-3), a binding protein of (+)-12-oxo-phytodienoic acid (OPDA), crisscrosses stress responses with light-dependent redox reactions, which fine-tunes the activity of key enzymes in the plastid photosynthetic carbon assimilation and sulfur assimilation pathways. Under stressed states, OPDA – accumulated in the chloroplasts – binds and promotes CYP20-3 to transfer electron (e−) from thioredoxins (i.e. type-f2 and -x) to 2-Cys peroxiredoxin B (2-CysPrxB) or serine acetyltransferase 1 (SAT1). Reduction (activation) of 2-CysPrxB then optimizes peroxide detoxification and carbon metabolisms in the photosynthesis, whereas the activation of SAT1 stimulates sulfur assimilation which in turn coordinates redox-resolved nucleus gene expressions in defense responses against biotic and abiotic stresses. Thus, we conclude that CYP20-3 is positioned as a unique metabolic hub in the interface between photosynthesis (light) and OPDA signaling, where controls resource (e−) allocations between plant growth and defense responses.

Polyploidy events are believed to be responsible for increasing the size of plant organs and enhancing tolerance to environmental stresses. Autotetraploid Paulownia australis plants exhibit superior traits compared with their diploid progenitors. Although some transcriptomics studies have been performed and some relevant genes have been revealed, the molecular and biological mechanisms regulating the predominant characteristics and the effects of polyploidy events on P. australis remain unknown. In this study, we compared the phenotypes, microstructures, and proteomes of autotetraploid and diploid P. australis plants. Compared with the diploid plant, the leaves of the autotetraploid plant were longer and wider, and the upper epidermis, lower epidermis, and palisade layer of the leaves were thicker, the leaf spongy parenchyma layer was thinner, the leaf cell size was bigger, and cell number was lower. In the proteome analysis, 3,010 proteins were identified and quantified, including 773 differentially abundant proteins. These results may help to characterize the P. australis proteome profile. Differentially abundant proteins related to cell division, glutathione metabolism, and the synthesis of cellulose, chlorophyll, and lignin were more abundant in the autotetraploid plants. These results will help to enhance the understanding of variations caused by polyploidy events in P. australis. The quantitative real-time PCR results provided details regarding the expression patterns of the proteins at mRNA level. We observed a limited correlation between transcript and protein levels. These observations may help to clarify the molecular basis for the predominant autotetraploid characteristics and be useful for plant breeding in the future.

Acetyl-CoA is an important precursor in Saccharomyces cerevisiae. Various approaches have been adopted to improve its cytosolic level previously with the emphasis on engineering the “acetyl-” part of acetyl-CoA. To the best of our knowledge, there have been no reports on engineering the “-CoA” part so far. In this study, we had tried to engineer S. cerevisiae from both the “-CoA” part via pantothenate kinase overexpression (PanK from S. cerevisiae, the rate-limiting enzyme for CoA synthesis) and the “acetyl-“part through PDH bypass introduction (ALD6 from S. cerevisiae and SeAcs L641P from Salmonella enteric). A naringenin-producing reporter strain had been constructed to reflect cytosolic acetyl-CoA level as acetyl-CoA is the precursor of naringenin. It was found that PanK overexpression or PDH bypass introduction alone only led to a twofold or 6.74-fold increase in naringenin titer, but the combination of both (strain CENFPAA01) had resulted in 24.4-fold increase as compared to the control (strain CENF09) in the presence of 0.5 mM substrate p-coumaric acid. The supplement of PanK substrate pantothenate resulted in another 19% increase in naringenin production. To greatly enhance acetyl-CoA level in yeast cytosol, it is feasible to engineer both the “acetyl-” part and the “-CoA” part simultaneously. Insufficient CoA supply might aggravate acetyl-CoA shortage and cause low yield of target product.

BACKGROUND: As a new protein expression and self-immobilization system, cell-surface displayed enzymes have attracted increasing attention. In this study, Geotrichum sp. lipase (GSL), an important enzyme for the enrichment of polyunsaturated fatty acids (PUFAs), was first displayed on the cell surface of Saccharomyces cerevisiae. RESULTS: The activity of displayed GSL was higher (43.7 U g−1 dry cell) than that of Candida antarctica lipase B (26.26 U g−1 dry cell) and that of Rhizopus oryzae lipase (4.1 U g−1 dry cell). It also exhibited higher thermostability than the free lipase, and retained 89% of the original activity after incubation at 40 °C for 3 h, compared with 48% at 35 °C for the free lipase at pH 8.5. Interestingly, the displayed lipase had a wider pH range and better pH stability. It had higher activity at all pH values than the free GSL, and retained 86% of the original activity in the pH range 9.5 to 10.5, whereas the activity of the free GSL could not be detected at pH 10. CONCLUSION: This work presented a method to prepare a whole-cell biocatalyst with better stability and broader pH tolerance which will provide a useful strategy for other cost-effective self-immobilized industrial lipases. Copyright © 2011 Society of Chemical Industry

Aspergillus niger lipase (ANL), a widely used hydrolase, was displayed for the first time on the surface of Saccharomyces cerevisiae using a-agglutinin as an anchor protein. Localization of ANL on the cell surface was confirmed by immunofluorescence microscopy. The displayed ANL was confirmed to be active toward tributyrin and p-nitrophenyl caprylate (pNPC). The hydrolytic activity toward pNPC reached 43.8 U/g of dry cell weight after induction by galactose for 72 h. The ANL-displaying cells were characterized for their use as whole-cell biocatalysts. The optimum temperature was 45 °C, and the pH was 7.0. The cells had good thermostability, retaining almost 80% of the full activity after incubation at 60 °C for 1 h, and >80% of the full activity at 50 °C for 6 h. The displayed lipase showed a preference for medium-chain fatty acid p-nitrophenyl esters. Therefore, the produced whole-cell catalyst is likely to have a wide range of applications.

Growth and reproduction of triploid and diploid blacklip abalone Haliotis rubra (Leach, 1814) were compared in a 30-month study. Triploidy was induced by inhibition of the second polar body formation using 6-dimethylaminopurine (6-DMAP) or cytochalasin B (CB). There were no significant differences in growth and survivorship between triploid and diploid abalone. However, triploid abalone had a more elongated shell and greater foot muscles than diploid abalone. A slightly curvilinear growth in shell length was conformed to all treatments. While diploid abalone had reached sexual maturity and spawned during the study, gonadal development and gamete maturation were abnormal in triploids. Female triploids lacked an apparent gonad at the macroscopic level but microscopic examination revealed that they had a thin layer of oogonia development. In contrast, male triploids were able to form similar-sized gonads to diploids during most of the reproductive period, but with brown-yellow discolouration and stalled gametogenesis at spermatocyte formation. Sex ratio of triploid abalone did not deviate from 1:1. With the onset of sexual maturation, growth and gonadal maturation occurred concurrently in diploid abalone, and there was no indication that growth of (diploid) abalone was reduced.

A key concern when developing artificial aquaculture diets is their palatability. This study investigated whether the palatability of artificial diets could be improved by incorporating a small quantity of highly preferred natural foods as feeding stimulants. The preference of Tripneustes gratilla for marine plants with which it co-occurs naturally in New South Wales, Australia, was assessed in a laboratory choice feeding experiment. T. gratilla displayed significant preference for the brown alga Ecklonia radiata over five other algae and one seagrass when they were offered simultaneously. Total protein or energy in the plants was not found to account for this preference. However, T. gratilla showed no preference among E. radiata, Sargassum linearifolium and Ulva lactuca when they were dried. Three artificial diets were made by incorporating one of each of these dried seaweeds at 5% dry weight. Although not statistically significant, T. gratilla ate more than twice as much the artificial diet containing S. linearifolium compared to the control diet containing no algae in a choice feeding experiment. In a no-choice feeding experiment, T. gratilla consumed significantly more of the Ecklonia and the Sargassum diets than the control diet despite each of the diets containing approximately the same protein and energy levels. T. gratilla consumed 49 and 43% more protein, and 37 and 44% more energy, respectively, when fed with the Ecklonia and the Sargassum diets compared to the control diet. Juvenile T. gratilla grew significantly faster on a wet weight basis on the Sargassum diet than the control diet at an average increase of 2.4% per day. These results indicate that the small amounts of palatable seaweed added to the artificial diets act as feeding stimulants, increasing the acceptability of artificial sea urchin diets, boosting the protein and energy consumption, and significantly increasing the growth of T. gratilla. Moreover, the fast growth of T. gratilla achieved in this study also indicates that this species would be a good candidate for commercial aquaculture.

The recently developed CRISPR/Cas9 system is a promising technology for targeted genome editing in a variety of species including plants. However, the first generation systems were designed to target one or two gene loci at a time. We designed a new multiplex CRISPR/Cas9 system that allows the co-expression of six sgRNA modules in one binary vector using a simple (three steps) cloning strategy in Arabidopsis. The transcription of the sgRNA modules is under the control of three different RNA Polymerase III-dependent promoters. We tested the efficiency of the new multiplex system by targeting six of the fourteen PYL families of ABA receptor genes in a single transformation experiment. One line with mutations in all six targeted PYLs was identified from 15 T1 plants. The mutagenesis frequency for the six individual PYL targets in the T1 lines ranged from 13 to 93 %. In the presence of ABA, the transgenic line identified as containing mutations in all six PYL genes produced the highest germination rate in the T2 progeny (37 %). Among these germinated seedlings, half of the analyzed plants (15/30) were homozygous mutants for at least four targeted genes and two plants (6.7 %) contained homozygous mutations in five of the targeted PYLs and the other targeted PYL had biallelic mutations. Homozygous sextuple mutants were identified in the T3 progeny and characterized together with previously described triple and sextuple PYL mutants. We anticipate that the application of this multiplex CRISPR/Cas9 system will strongly facilitate functional analysis of genes pathways and families.

DNA methylation is a conserved epigenetic mark that plays important roles in plant and vertebrate development, genome stability, and gene regulation. Canonical Methyl-CpG-binding domain (MBD) proteins are important interpreters of DNA methylation that recognize methylated CG sites and recruit chromatin remodelers, histone deacetylases, and histone methyltransferases to repress transcription. Here, we show that Arabidopsis MBD7 and Increased DNA Methylation 3 (IDM3) are anti-silencing factors that prevent gene repression and DNA hypermethylation. MBD7 preferentially binds to highly methylated, CG-dense regions and physically associates with other anti-silencing factors, including the histone acetyltransferase IDM1 and the alpha-crystallin domain proteins IDM2 and IDM3. IDM1 and IDM2 were previously shown to facilitate active DNA demethylation by the 5-methylcytosine DNA glycosylase/lyase ROS1. Thus, MBD7 tethers the IDM proteins to methylated DNA, which enables the function of DNA demethylases that in turn limit DNA methylation and prevent transcriptional gene silencing.

Optimal conditions of 6-dimethylaminopurine (6-DMAP) for triploidy induction in the blacklip abalone Haliotis rubra (Leach, 1814) were investigated, targeting inhibition of second polar body (PB2) formation. Two experiments were conducted at a water temperature of 17.5–18.5°C where factorial combination of (1) four dosages (DSs) of 100, 150, 200 and 250 μM 6-DMAP, four starting times (STs) of 15, 20, 25 and 30 min post fertilization, and two treatment durations (TDs) of 20 and 30 min and (2) three DSs of 50, 100 and 150 μM 6-DMAP, three STs of 15, 20 and 25 min post fertilization, and three TDs of 10, 20 and 30 min, were applied respectively. Day 3 larvae were sampled for triploidy and survival. Percent triploidy was verified using flow cytometry (FCM). Results show that optimal inductions that combine both high rates of triploidy and reasonable survival were those treatments commenced 15 or 20 min post fertilization and continued for 20 or 30 min, using 100 μM 6-DMAP. These conditions induced rates of triploidy and relative survival of 80.5–93.3% and 36.5–40.2%, respectively, in the first experiment, and corresponding rates were 79.1–93.6% and 20.7–43.0% in the second experiment. High percent triploidy were also obtained in a number of treatments using 150 μM 6-DMAP, but with overall survival rates generally lower than those using 100 μM 6-DMAP.

Large batches of sibling triploid and diploid juvenile Haliotis rubra were produced to evaluate and compare their production performances. Triploidy was induced by inhibiting extrusion of the second polar body (PB2) using either 150 μM 6-dimethylaminopurine (6-DMAP) or 0.5 mg/l cytochalasin B (CB). Best guess conditions were used in which treatments were started at 25–26 min after fertilization and continued for 20 min at 17.5–18.5 °C. Triploid percentages, survival and growth were examined up to 22 weeks after settlement. Triploid percentages in day 2 larvae were 94% and 92% in 6-DMAP and CB treatments, respectively. Overall survival from eggs to competent larvae was 30.0 and 55.5% for 6-DMAP and CB treatments, respectively, compared to 64.8% for the diploids. Yields of 20 weeks post-settlement juveniles (from competent larvae) were less than 1.9% in the two triploid treatments and the diploid control. A shell length was less than 3.7 mm in all the three cases. At 22 weeks after settlement, triploid rates were 100% for 6-DMAP and 82.5% for CB treatments.

This paper describes the culture of Venus clam larvae, Cyclina sinensis (Gmelin), to metamorphosis and the first successful case of rearing Venus clam seeds on a commercial scale and at high density. Performances of broodstock, eggs and larvae were examined. Metamorphosed larvae (seeds) produced were 2.3×10 8 in a rearing tank of 31.8 m 2 in base at the end of the experiment, with a mean shell length×shell height of 259.6×242.0 μm, giving a production of 7.23×10 6 seeds/m 2 .

Paulownia witches’ broom (PaWB) disease caused by phytoplasma is a fatal disease that leads to considerable economic losses. Although there are a few reports describing studies of PaWB pathogenesis, the molecular mechanisms underlying phytoplasma pathogenicity in Paulownia trees remain uncharacterized. In this study, after building a transcriptome database containing 67,177 sequences, we used isobaric tags for relative and absolute quantification (iTRAQ) to quantify and analyze the proteome-level changes among healthyP. fortunei(PF), PaWB-infectedP. fortunei(PFI), and PaWB-infectedP. fortuneitreated with 20 mg L−1or 60 mg L−1methyl methane sulfonate (MMS) (PFI-20 and PFI-60, respectively). A total of 2,358 proteins were identified. We investigated the proteins profiles in PFvs. PFI (infected process) and PFI-20vs. PFI-60 (recovered process), and further found that many of the MMS-response proteins mapped to “photosynthesis” and “ribosome” pathways. Based on our comparison scheme, 36 PaWB-related proteins were revealed. Among them, 32 proteins were classified into three functional groups: (1) carbohydrate and energy metabolism, (2) protein synthesis and degradation, and (3) stress resistance. We then investigated the PaWB-related proteins involved in the infected and recovered processes, and discovered that carbohydrate and energy metabolism was inhibited, and protein synthesis and degradation decreased, as the plant responded to PaWB. Our observations may be useful for characterizing the proteome-level changes that occur at different stages of PaWB disease. The data generated in this study may serve as a valuable resource for elucidating the pathogenesis of PaWB disease during phytoplasma infection and recovery stages.

Lipase has been used widely in industry. In this study, we have constructed two recombinant Saccharomyces cerevisiae strains displaying two active lipases on the cell surface by cell surface engineering. The genes encoding Yarrowia lipolytica lipases Lip7 and Lip8 were fused with the gene encoding small binding subunit Aga2 of a-agglutinin. Localization of the Lip7 and Lip8 on the cell surface was confirmed by immunofluorescence microscopy. Besides, the putative signal sequences of Lip7 and Lip8 were removed to compare their effect on the activities of surface-displayed lipases. The results showed that the activities towards p-nitrophenyl caprylate of surface-displayed Lip7 and Lip8 were 283 U/g (dry cell) and 121 U/g (dry cell), much higher than that using Flo1 as anchor protein in Pichia pastoris, and the putative signal sequences have significant effect on the activities of the displayed lipases; when deleted, the lipases’ activities were declined to 65 U/g (dry cell) and 80 U/g (dry cell), respectively. The displayed lipases exhibit a preference for middle chain fatty acids and a high thermal stability. Additionally, from the study, to surface-display a target protein, it is recommendable that the structure feature of the protein should be assayed through bioinformatics methods and then the cell wall proteins with the anchor domain far away from the activity center should be chosen as anchor proteins.

Red recombinase system of the lambda phage is widely used for recombination of short linear DNA fragments and genome. Using this system, we obtained T7 RNA polymerase (RNAP) substitution mutants in Burkholderia cepacia. To test the expression abilities of the T7 mutants, four different lipase expression vectors were transformed and the lipase activity of these recombinants was evaluated. Our results suggest that 500 nt homology between the unit and the genome is sufficient to generate mutations and this strategy enables the rapid establishment of mutant strains with efficiencies of 85%. After expression and purification, the highest purified lipase activity obtained was 3,990 U/l, nearly triple that of the wild-type organism.

Lipase Lip2 from Yarrowia lipolytica was displayed on the cell surface of Saccharomyces cerevisiae using Cwp2 as an anchor protein. Successful display of the lipase on the cell surface was confirmed by immunofluorescence microscopy and halo assay. The length of linker sequences was further examined to confirm that the correct conformation of Lip2 was maintained. The results showed that the displayed Lip2 exhibited the highest activity at 7.6 +/- 0.4 U/g (dry cell) when using (G(4)S)(3) sequence as the linker, with an optimal temperature and pH at 40 degrees C and pH 8.0. The displayed lipase did not lose any activity after being treated with 0.1% Triton X-100 and 0.1% Tween 80 for 30 min, and it retained 92% of its original activity after incubation in 10% DMSO for 30 min. It also exhibited better thermostability than free Lip2 as reported previously.