A simple and efficient CRISPR/Cas9 platform for induction of single and multiple,heritable mutations in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Background

Genome editing of monocot plants can be accomplished by using the components of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9) technology specifically optimized for these types of plants. Here, we present the development of RNA-guided Cas9 system for simplex and multiplex genome editing in barley.

Results

We developed a set of customizable RNA-guided Cas9 binary vectors and sgRNA modules for simplex and multiplex editing in barley. To facilitate the design of RNA-guided Cas9 constructs, the pBract derived binary vectors were adapted to Gateway cloning and only one restriction enzyme was required for construction of the sgRNA. We designed a synthetic, codon optimized Cas9 gene containing the N terminal SV40 nuclear localization signal and the UBQ10 Arabidopsis 1st intron. Two different sgRNAs were constructed for simplex editing and one polycistronic tRNA-gRNA construct (PTG) for multiplex editing using an endogenous tRNA processing system. The RNA-guided Cas9 constructs were validated in transgenic barley plants produced by Agrobacterium-mediated transformation. The highest mutation rate was observed in simplex editing of the cytokinin oxidase/dehydrogenase HvCKX1 gene, where mutations at the hvckx1 locus were detected in 88% of the screened T₀ plants. We also proved the efficacy of the PTG construct in the multiplex editing of two CKX genes by obtaining 9 plants (21% of all edited plants) with mutations induced in both HvCKX1 and HvCKX3. Analysis of the T₁ lines revealed that mutations in the HvCKX1 gene were transmitted to the next generation of plants. Among 220 screened T₁ plants we identified 85 heterozygous and 28 homozygous mutants, most of them bearing frameshift mutations in the HvCKX1 gene. We also observed independent segregation of mutations and the Cas9-sgRNA T-DNA insert in several T₁ plants. Moreover, the knockout mutations of the Nud gene generated phenotype mutants with naked grains, and the phenotypic changes were identifiable in T₀ plants.

Conclusions

We demonstrated the effectiveness of an optimized RNA-guided Cas9 system that can be used for generating homozygous knockout mutants in the progeny of transgenic barely plants. This is also the first report of successful multiplex editing in barley using a tRNA processing system.

     

Effects Of Thinning In a Water-Limited Holm Oak Forest

ABSTRACT

A natural holm oak forest was selectively thinned to test thinning as a tool to reduce tree mortality, increase productivity, and reverse the recent regression of the dominant species (Quercus ilex) induced by climate change. Thinning increased aboveground productivity and reduced stem mortality in this Mediterranean forest during four years just after thinning, contributing to the maintenance of forest functioning under changing climatic conditions. Q. ilex was the only species positively affected by the thinning: stem growth increased for all stem sizes, and mortality was significantly lower in thinned plots. On the contrary, mortality rates of Phillyrea latifolia and Arbutus unedo were not significantly lower. Stem growth increased for P. latifolia only in the smallest stem-size class. Our results highlight the suitability of selective thinning for improving the forest productivity and ensuring the conservation of Mediterranean coppices. Other benefits of selective thinning, such as a decrease in the risk of fire dispersion and an increase in the water supply for human populations, are also discussed.     

Valuing forest recreation on the national level in a transition economy: The case of Poland

Recreation benefits constitute a substantial part of the total economic value of forests, and are important for the choice of multi-functional forest policies. The application of methods valuing such benefits is in its infancy in transition economies in Central and Eastern Europe (CEE), so value estimates for policy use are sometimes transferred from Western Europe proportionally scaled down by GDP. However, little is known about how recreation values vary with income, and one risks underestimating benefits in CEE. This paper reports the findings of the first comprehensive, national-level study in any CEE country estimating annual and per trip forest recreation values in Poland using the Travel Cost (TC) and Contingent Valuation (CV) methods. Two in-person interview surveys of forest recreation behaviour were carried out. The first was administered on-site in ten representative forest areas, and the other in the homes of a national sample of adult Poles. Results show that forest recreation is highly valued in Poland, at Euros 0.64–6.93 per trip per person, depending on the valuation method. Both trip frequency and per trip values are higher than the average in Western Europe, despite a lower income level. Thus, a simple GDP-adjusted transfer from Western Europe would substantially undervalue forest recreation in Poland. Further, a comparison of TC consumer surplus estimates and GDP/capita in Europe shows no clear relationship, indicating that a range of cultural, institutional and other factors may be important.     

Study of dynamic accumulation of secondary metabolites in three subspecies of Hypericum perforatum

St. John's Wort extracts are used for the treatment of mild to moderately severe depression, and their composition and standardization have been thoroughly investigated. Standardization of St. John's Wort extracts has to cope with several factors that affect the phytochemical profile of the plant, with geographic location, seasonal variations and subspecies all being important. To address this issue, three different subspecies of Hypericum perforatum L. were profiled in relation to different maturation phases, evaluating the variations in the context of the major secondary metabolites from this plant. HPLC analysis indicated that H. perforatum subsp. perforatum is richer in secondary metabolites than the other subspecies, and that the three subspecies show a different profile during the developmental stages. Hypericins, hyperforins and flavonoids peak at quite different stages, and the standardization of Hyperici extracts based only on the contents of hypericin is not adequate to guarantee batch reproducibility of the extracts.     

Mechanisms and metabolomics of the host–pathogen interactions between Chestnut (Castanea species) and Chestnut blight (Cryphonectria parasitica)

Chestnut blight is a stem‐girdling disease of Castanea caused by the fungal pathogen Cryphonectria parasitica. Chestnut blight affects all Castanea species to some degree. In Asian species, chestnut blight is a commercially relevant disease which primarily affects nut production. In American and European species, chestnut blight has caused significant declines in wild populations and continues to negatively affect nut production in the European chestnut (C. sativa). Despite the profound effect of this disease in the Castanea genus, very little is known concerning the factors involved in the host–pathogen interaction between C. parasitica and its Castanea hosts. This review summarizes information on known mechanisms and metabolites involved in the host–pathogen interaction and contributes original information on the pathogen in relation to susceptible and putatively resistant genotypes with a view to furthering research that will promote a better understanding of this devastating disease and enable its control.     

Breeding bird response to partially harvested riparian management zones

We compared avian communities among three timber harvesting treatments in 45-m wide even-age riparian management zones (RMZs) placed between upland clearcuts and along one side of first- or second-order streams in northern Minnesota, USA. The RMZs had three treatments: (1) unharvested, (2) intermediate residual basal area (RBA) (targeted goal 11.5 m²/ha, realized 16.0 m²/ha), and (3) low RBA (targeted goal 5.7 m²/ha, realized 8.7 m²/ha). Surveys were conducted one year pre-harvest and three consecutive years post-harvest. There was no change in species richness, diversity, or total abundance associated with harvest but there were shifts in the types of birds within the community. In particular, White-throated Sparrows (Zonotrichia albicollis) and Chestnut-sided Warblers (Dendroica pensylvanica) increased while Ovenbirds (Seiurus aurocapilla) and Red-eyed Vireos (Vireo olivaceus) decreased. The decline of avian species associated with mature forest in the partially harvested treatments relative to controls indicates that maintaining an unharvested RMZ adjacent to an upland harvest may aid in maintaining avian species associated mature forest in Minnesota for at least three years post-harvest. However, our observations do not reflect reproductive success, which is an area for future research.     

Trade-offs and synergies in management of two co-occurring specialist squash pests

Journal of Pest Science - Co-occurring herbivorous pests may have shared or divergent responses to plant- and insect- derived cues, creating challenges for effective pest management in...     

Mid-infrared absorption properties of green wood

There is a lack of quantitative data on the penetration depth and the amount of energy absorbed by green wood under infrared (IR) radiation. This lack of knowledge is a potential barrier to the development of IR heating as an alternative to soaking as a means of warming logs prior to peeling in the manufacture of plywood. Experimental measurements of normal hemispherical spectral reflectance and transmittance over the range 550–5,500 cm^?1 wavenumbers on four wood species, beech, birch, Douglas-fir and spruce have brought new knowledge on mid-infrared absorption properties of green wood and removed some uncertainties. For instance, it is not possible to deliver energy deeper than up to 0.3 mm below the wood surface because 70–90 % of all incident IR radiation on the wood surface is absorbed in this layer. Some wood features, such as surface quality, the presence of knots and of free water in wood (the latter two having a more significant effect) influence the amount of energy absorbed. These results illustrate that IR radiation can heat the surface layers, but then heat penetrates deeper into the inside layers of wood by conduction.     

A novel extracellular peroxidase and nucleases from a milky sap of Chelidonium majus

Using affinity chromatography, SDS-PAGE, peroxidase activity assay and mass spectrometry data, a new extracellular peroxidase (CMP) from Chelidonium majus milky sap was isolated and characterized. The protein has a molecular weight of about 40 kDa and belongs to secretory class III plant peroxidases. The peroxidase activity is also accompanied by DN-ase activities. A novel CMP combined with other proteins is probably involved in development and differentiation of the plant and defence against different pathogens. It suggests that the biological activity of C. majus whole plants and extracts may depend not only on its alkaloidal content but also on the presence of biologically active proteins.