Comparison of Organic and Chemical Soil Amendments Used in the Reforestation of a Harsh Sierra Nevada Site

Abstract A comparison of a composted organic amendment, a controlled‐release fertilizer, and induced mycorrhizal inoculation as affecting the establishment and nutrition of bareroot Jeffrey pine (Pinus jeffreyi Grev. & Balf.) was conducted on a Sierra Nevada surface mine. The soil amendments were applied at outplanting to the backfill of augered planting holes, with a low rate of 8 g and a high rate of 16 g per seedling for the fertilizer, Gromax 21‐6‐2 + Minors, whereas a single rate of 2.0 L was used for organic matter. Colonization by Pisolithus tinctorius (Pers.) Coker & Couch was induced by coating the root systems with basidiospores suspended in a gel carrier. The organic amendment especially, but also mycorrhizal inoculation, caused substantial seedling mortality, whereas survival was unaffected by controlled‐release fertilization. Gromax applied at the high rate produced a 74% increase in shoot volume after three growing seasons, whereas the organic amendment reduced volume by 28%. Growth was unaffected by mycorrhizal treatment. The growth response to the 16‐g Gromax application probably reflected enhanced N, P, and K nutrition and decreased concentrations of potentially toxic metallic elements, including Mn and Al among others, as revealed through foliar analysis. Because they were accompanied by growth reduction, nutritional responses to the organic amendment, which involved both macronutrients and trace elements, were of little consequence. Impaired water relations may account for the poor response to this amendment. Likewise, nutritional responses to mycorrhizal inoculation produced no discernible benefit in terms of seedling performance. An inoculation procedure that failed to induce substantially greater P. tinctorius colonization in inoculated than uninoculated seedlings, and that may have also impaired water relations, likely explains this result. Overall, these findings indicate that further research is needed before either the organic amendment or the mycorrhizal inoculation procedure used here can be used in forest restoration efforts on dry sites.     

<i>In Vitro</i>-Propagation of <i>Agave tequilana</i> Weber cv. azul in a Temporary Immersion System

In Mexico, there is a need to produce large quantities of plantlets for the establishment and replanting of blue (cv. azul) agave production areas. Most of these plots are within the origin denomination area (DOT, Spanish acronym) of the distilled product of this plant, known as tequila. The objective of this study was to develop an in vitro-propagation protocol for Agave tequilana Weber cv. azul using segmented stems in both: solid and liquid media. A disinfection and in vitro technique were developed to obtain shoots, through plantlets collected in commercial plots, which attained 100% surface-disinfection and budding rate. At the multiplication stage, the effects of 6-Benzylaminopurine (BA) (0.0, 4.4 and 13.2 μM) and kinetin (0.0, 9.4, 18.8 and 37.6 μM) were evaluated on lateral-shoot production of segmented sagittal stems. These were cultivated on Murashige & Skoog (MS) medium, with the addition of 3.0% sucrose and 8 g L⁻¹ agar. It was observed that BA and kinetin increased the number of shoots per explant, obtaining up to 18 and 26, respectively. Furthermore, it was found that just the sagittal segmentation of explants increased axillary budding. On the other hand, segmented-stem bases were grown in MS liquid medium with 3.0% sucrose, inside a RITA® system, programmed by a 5 min immersion step with a frequency of every 4 h. The effect of Indole−3-Acetic acid (IAA) (0.57, 2.9, 5.7 μM) was evaluated, while maintaining a concentration of BA (13.2 μM). It was observed that the greatest concentration of IAA led to the formation of more than 20 buds per explant. These results offer a new methodology to increase the efficiency of A. tequilana Weber cv. azul-in vitro multiplication by sagittal segmentation of stems and the addition of BA and/or IAA.     

Engineering mammalian mucin-type O-glycosylation in plants

Mucin-type O-glycosylation is an important post-translational modification that confers a variety of biological properties and functions to proteins. This post-translational modification has a particularly complex and differentially regulated biosynthesis rendering prediction and control of where O-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity. Transient expression of a Pseudomonas aeruginosa Glc(NAc) C4-epimerase and a human polypeptide GalNAc-transferase in leaves of Nicotiana benthamiana resulted in GalNAc O-glycosylation of co-expressed human O-glycoprotein substrates. A chimeric YFP construct containing a 3.5 tandem repeat sequence of MUC1 was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon α2b. In plants, prolines in certain classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host cell for production of recombinant O-glycoproteins with custom-designed O-glycosylation. The observed hydroxyproline modifications, however, call for additional future engineering efforts.     

Morphogenesis of active shells

We consider the active shell as a single-cell or epithelial sheet surface that, sharing basic properties of stretched elastic shells, is capable of active planar movement owing to recruiting of the new surface elements. As model examples of their morphogenesis, we consider the growth and differentiation of single-cell hairs (trichomes) in plants of the genus Draba, and the epiboly and formation of the dorsoventral polarity in loach. The essential feature of the active shell behavior at both cellular and supracellular levels is regular deviating from the spatially homogeneous form, which is a primary cause of originating of the active mechanical stresses inside the shell in addition to its passive stretching by the intrinsic forces. Analyzing the quantitative morphological data, we derive the equations in which the temporal self-oscillations and spatial differentiation are distinguishable only at the parametric level depending on the proportion of active to passive stresses. In contrast to the ordinary activator-inhibitor systems, the self-oscillation dynamics is principally non-local and, consequently, one-parametric, the shell surface curvature being an analog of the inhibitor, while its spatial variance being an analog of the activator of shaping. Analyzing variability and evolution of the hair cell branching, we argue that the linear ontogeny (succession of the developmental stages) is a secondary evolutionary phenomenon originating from cyclic self-organizing algorithms of the active shell shaping.     

Xanthophylls are key constituents of semi‐synthetic diets for continuous breeding of Pieris brassicae without food plant

Semi‐synthetic or fully artificial diets are well established in culturing laboratory insects on a large scale. In Pieris brassicae L. (Lepidoptera: Pieridae), a published semi‐synthetic diet supplemented with sinigrin, a chemical label of cruciferous plants, is well accepted by the larvae but the adults fail to produce offspring; continuous breeding, therefore, requires the addition of cabbage powder to the diet. We set out to identify essential substances to eliminate the need for cabbage in our long‐term mass culture. The studies revealed that the dietary difference, which becomes most evident at the adult stage only, is because of a severe lack of carotenoids in the cabbage‐free version. This was uncovered in larvae, fed cabbage‐supplemented diet, that respond to blue light of sufficient intensity by high cuticle melanization on pupation; larvae fed the carotenoid‐deficient sinigrin‐supplemented diet fail to respond in this way. Males were found to be more sensitive than females to blue light in this respect. The essential substance mediating this photo response was found to be lutein and/or zeaxanthin, two abundant xanthophylls of green leaves. β‐Carotene was only marginally active. These xanthophylls are known to serve as precursors to the synthesis of 3‐hydroxyretinal, the exclusive visual chromophore of lepidopteran insects; retinal, derived from β‐carotene, is not (substantially) used by these insects. In conclusion, the absence of flying and mating activity in the adults from the sinigrin diet may indicate visual deficiency owing to a lack of a specific structural class of carotenoids in the larval food. Our results enabled us to formulate a semi‐synthetic diet that allows continuous mass rearing of P. brassicae without cabbage at any stage including oviposition. In addition to the key constituents sinigrin and xanthophylls, this superior diet includes tryptophan, as methyl ester, and α‐tocopherol.     

The inhibition of infection thread development in the cultivar-specific interaction ofRhizobium and subterranean clover is not caused by a hypersensitive response

Summary The cultivar specific interaction ofTrifolium subterranean cv. Woogenellup andRhizobium leguminosarum bv.trifolii strain ANU 794 was examined to establish the basis for nodulation failure on this cultivar. Infections were initiated by strain ANU 794 on cv. Woogenellup. Root hair curling, the initiation of infection threads, and cortical cell divisions were evident on the tap root and appeared normal after microscopic observation. However, in most cases, the infection threads stayed confined to the root hairs. No evidence was found for a hypersensitive response by the plant. The progress of infections on the tap roots was different from that on the lateral roots. This was confirmed by the differential tap and lateral root nodulation patterns of the mutants derived from strain ANU 794, which show enhanced nodulation on cv. Woogenellup. On the lateral roots, cortical cell divisions progressed further than those on the tap root and formed macroscopically visible swellings, which could be divided into two morphological classes. In some cases infection threads developed into these primordia but successful nodules were not established. The inhibition of infection appeared to be manifested at two levels: first, on the tap roots in the root hairs, where many of the infection threads are contained and secondly, in the primordia induced on the lateral roots, where the infection threads sometimes penetrate further than the root hair cell but stop in the primordial cells. It appears that an essential factor or trigger in the communication between plant and bacteria is missing or altered, resulting in an array of primordia-structures, which cease to develop.Abbreviations bv biovar - cv cultivar - Fix⁺ nitrogen fixing - GUS -glucuronidase - Nod⁺ nodulating - HR hypersensitive response - Km kanamycin - LOSs lipo-oligosaccharides - Sm streptomycin - Sp spectinomycin - X-Gluc 5-bromo-4-chloro-3-indonyl--glucuronic acid     

Human impact during the Bronze Age on the vegetation at Lago Lucone (northern Italy)

Lake-sediment records were used to reconstruct human impact on the landscape around Lago Lucone (45°33′N, 10°29′E, 249 m a.s.l.), a former lake in the western amphitheatre system of the Lago di Garda. Presence of prehistoric human populations is attested by pile-dwelling settlements from the Early-Middle Bronze Age, with one settlement at a distance of only 100 m from the coring site. Pollen, plant-macrofossil and microscopic charcoal analyses were applied to a 250 cm sediment core with four dates providing the time control. A mixed oak forest that was important during the Early-Middle Holocene was cleared and replaced by open vegetation during the Bronze Age (∼2000–1100 b.c.) when open lands were estimated to have covered more than 60% of the total relevant pollen-source area. During a phase of high human impact, independent climatic proxies suggest warm and dry climatic conditions. Later, ca. 1100 b.c., palaeobotanical evidence indicates a sharp decrease in human pressure in the Lago Lucone area. The comparison with other sedimentary palaeocultural records shows that the period 1300–1100 b.c. was characterised by general declines of agricultural activities both south and north of the Alps. These declines have been previously attributed to a change towards wetter and colder climatic conditions in and around the Alps. However, the decline in human impact around Lago Lucone cannot be exclusively attributed to climatic variation. Therefore other forcing factors independent of climatic changes, such as cultural crises or changes in spatial organisation of the habitats, cannot be ruled out under the present state of knowledge.     

A new technique for studying the stylet tracks of homopteran insects in hand-sectioned plant tissue using light or epifluorescence microscopy

Homopteran insects, such as aphids, psyllids and scales, inject a proteinaceous salivary sheath into their host plant tissue during feeding. This sheath, also referred to as a stylet track, remains in the tissue after the stylets are withdrawn, and is useful for studying plant resistance to insects and plant virus transmission. We describe a new method for studying stylet tracks. Hand microtome sectioned plant material was fixed and cleared in ethanol. The stylet tracks were stained with acid fuchsin and counterstained with aniline blue or fast green. The acid fuchsin stained stylet tracks were pink to red under light microscopy, and orange under TRITC epifluorescence. Stylet tracks in unstained sections autofluoresced under DAPI epifluorescence. This new technique is significantly faster and less complex than previous techniques, and permitted visualization of stylet tracks with light or epifluorescence microscopy within 1 hr of collecting fresh plant material. The technique was also applicable to a broad range of homopterans and plant taxa and provided excellent photomicrographs.     

Characterization of two distinct feruloyl esterases, AoFaeB and AoFaeC, from Aspergillus oryzae

Two hypothetical proteins XP_001818628 and XP_001819091 (designated AoFaeB and AoFaeC, respectively), showing sequence identity with known type-C feruloyl esterases, have been found in the genomic sequence of Aspergillus oryzae. We cloned the putative A. oryzae feruloyl esterase-encoding genes and expressed them in Pichia pastoris. Both purified recombinant AoFaeB (rAoFaeB) and AoFaeC (rAoFaeC) had apparent relative molecular masses of 61,000 and 75,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After N-deglycosylation, both proteins had a relative molecular mass of 55,000. The optimum pH for rAoFaeB was 6.0, although it was stable at pH values ranging from 3.0 to 9.0; rAoFaeC had an optimum pH of 6.0 and was stable in the pH range of 7.0–10.0. Thermostability of rAoFaeC was greater than that of rAoFaeB. Whereas rAoFaeC displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, methyl ferulate, and methyl sinapate, rAoFaeB displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, and methyl ferulate but not toward methyl sinapate. Substrate specificity profiling of rAoFaeB and rAoFaeC revealed type-B and type-C feruloyl esterases, respectively. Ferulic acid was efficiently released from wheat arabinoxylan when both esterases were applied with xylanase from Thermomyces lanuginosus. Both recombinant proteins also exhibited hydrolytic activity toward chlorogenic acid. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.