A selection strategy in plant transformation based on antisense oligodeoxynucleotide inhibition

Antisense oligodeoxynucleotide (asODN) inhibition was developed in the 1970s, and since then has been widely used in animal research. However, in plant biology, the method has had limited application because plant cell walls significantly block efficient uptake of asODN to plant cells. Recently, we have found that asODN uptake is enhanced in a sugar solution. The method has promise for many applications, such as a rapid alternative to time‐consuming transgenic studies, and high potential for studying gene functionality in intact plants and multiple plant species, with particular advantages in evaluating the roles of multiple gene family members. Generation of transgenic plants relies on the ability to select transformed cells. This screening process is based on co‐introduction of marker genes into the plant cell together with a gene of interest. Currently, the most common marker genes are those that confer antibiotic or herbicide resistance. The possibility that traits introduced by selectable marker genes in transgenic field crops may be transferred horizontally is of major public concern. Marker genes that increase use of antibiotics and herbicides may increase development of antibiotic‐resistant bacterial strains or contribute to weed resistance. Here, we describe a method for selection of transformed plant cells based on asODN inhibition. The method enables selective and high‐throughput screening for transformed cells without conferring new traits or functions to the transgenic plants. Due to their high binding specificity, asODNs may also find applications as plant‐specific DNA herbicides.     

Field of Dreams: Restitution of Pollinator Services in Restored Bird‐Pollinated Plant Populations

Ecosystem functionality is an increasingly important objective of ecological restoration. Despite this, a few studies have rigorously assessed reproductive functionality within restored plant populations, and it is largely assumed that pollinators follow restoration of plant communities—“build it and they will come.” Here, we applied an ecological genetic approach to determine the impact of spatial separation on mating in Banksia menziesii (Proteaceae), a dominant bird‐pollinated species of Banksia woodlands of Western Australia. All plants at three post‐mining restored sites (n = 72 [13 years old], n = 21 [8 years old], and n = 20 [9 years old]), as well as a sample from an adjacent natural reference site (n = 42), were genotyped at nine microsatellite loci. Seed set, mating system parameters, realized pollen dispersal through the assignment of paternity to seed, and avian pollinator species composition, abundance and behavior, were assessed. All patches displayed equivalent heterozygosity (H_e = 0.53–0.59) and very weak genetic divergence (F_ST ≤ 0.01). Seed of plants within restored sites showed complete outcrossing and relatively high seed set, 26% of which were sired by pollen donors located beyond the local patch. Similar abundance and movement of nectar‐feeding birds was observed in restored and natural sites, despite lower bird species diversity in the restored site, where a smaller, less aggressive species was dominant. Our results demonstrate the restitution of wide outcrossing in these restored Banksia patches within an active mine‐site, and suggest that restored bird‐pollinated Banksia populations are resilient to human impacts, due largely to their generalist pollinator requirements and highly‐mobile avian pollinators.     

The 60S associated ribosome biogenesis factor LSG1‐2 is required for 40S maturation in Arabidopsis thaliana

Ribosome biogenesis involves a large ensemble of trans‐acting factors, which catalyse rRNA processing, ribosomal protein association and ribosomal subunit assembly. The circularly permuted GTPase Lsg1 is such a ribosome biogenesis factor, which is involved in maturation of the pre‐60S ribosomal subunit in yeast. We identified two orthologues of Lsg1 in Arabidopsis thaliana. Both proteins differ in their C‐terminus, which is highly charged in atLSG1‐2 but missing in atLSG1‐1. This C‐terminus of atLSG1‐2 contains a functional nuclear localization signal in a part of the protein that also targets atLSG1‐2 to the nucleolus. Furthermore, only atLSG1‐2 is physically associated with ribosomes suggesting its function in ribosome biogenesis. Homozygous T‐DNA insertion lines are viable for both LSG1 orthologues. In plants lacking atLSG1‐2 18S rRNA precursors accumulate and a 20S pre‐rRNA is detected, while the amount of pre‐rRNAs that lead to the 25S and 5.8S rRNA is not changed. Thus, our results suggest that pre‐60S subunit maturation is important for the final steps of pre‐40S maturation in plants. In addition, the lsg1‐2 mutants show severe developmental defects, including triple cotyledons and upward curled leaves, which link ribosome biogenesis to early plant and leaf development.     

Can biodiversity hotspots protect more than tropical forest plants and vertebrates?

Conservation International's biodiversity hotspots are areas of high vascular plant endemism combined with high levels of habitat destruction and land use change. Although such hotspots have also been shown to be centres for terrestrial vertebrate endemism, much less is known about how well these areas function as hotspots for other less well‐studied groups, including the hyperdiverse arthropods, other invertebrates and fungi. Because there is a close evolutionary and ecological relationship between insects and plants, we suggest that the potential role of plants as umbrella species for herbivorous insects, potentially herbivorous fungi and nematodes, and parasitic insects should be explored. Finally, we reflect on the increasing social, economic, human conflict and governance issues and the impacts of increasing land use change and global climate change that threaten the biodiversity hotspot system.     

AMF和PGPR联合修复菲和芘污染土壤的效应

丛枝菌根真菌（arbuscular mycorrhizal fungi,AMF）与根围促生细菌（plant growth-promoting rhizobacteria,PGPR）联合降解有毒有机物、修复污染土壤和促进植物生长的作用倍受关注。本试验旨在探究AMF与PGPR联合降解土壤中菲和芘的效应,以菲和芘1:1混合处理浓度各0、50mg/kg、100mg/kg和150mg/kg下对高羊茅Festuca elata接种AMF根内根孢囊霉Rhizophagus intraradices（Ri）、变形球囊霉Glomus versiforme（Gv）、PGPR荧光假单胞菌Pseudomonas fluorescens Ps2-6、芽孢杆菌Bacillus velezensis Ps3-2、Ri+Ps2-6、Ri+Ps3-2、Gv+Ps2-6、Gv+Ps3-2和不接种对照共36个处理。结果表明,供试AMF增加了PGPR的定殖数量;接种PGPR则显著提高AMF的侵染率。AMF、PGPR或AMF+PGPR处理均显著降低土壤中菲和芘含量,促进植物对土壤中菲和芘的吸收,显著提高高羊茅根系和叶片内的菲和芘含量。在土壤中菲和芘100mg/kg和150mg/kg水平下,Gv与Ps2-6及Ri与Ps2-6能相互促进对土壤中菲和芘的去除效应,其中接种Gv+Ps2-6组合处理的去除率最高,达到95%-98%,土壤中多酚氧化酶、脱氢酶和过氧化氢酶活性显著高于单接种处理和不接种对照,而酸性磷酸酶活性变化则表现为相反趋势。其中以Gv+Ps2-6组合处理的多酚氧化酶活性最高,为0.17mg/g,是不接种对照的1.9倍;脱氢酶和过氧化氢酶活性分别达到1.32µg/(g·h)和1.81mL/g;酸性磷酸酶活性则比不接种对照土壤降低27%-45%;易提取球囊霉素相关土壤蛋白含量和总球囊霉素相关土壤蛋白含量分别是不接种对照的1.6倍和1.5倍。     

遮荫对凤眼莲生长及其生理适应机制

2011年于南京对凤眼莲进行不同光处理(全天遮荫、7:00～11:00遮荫、11:00～15:00遮荫和15:00～19:00遮荫),遮荫下光强为自然光强的70%,以全自然光为对照(CK),处理25d,每5d分别测定生长指标、单株鲜重、单株干重、光合净光合速率(Pn)、抗氧化酶、非酶系统的总抗氧化能力(AOC)以及1,5-二磷酸核酮糖羧化加氧酶(Rubisco)活性等生理指标,以阐明光强对其生长以及适应机制,为提高凤眼莲生物量提供依据。结果显示:与CK相比,全天遮荫明显降低了叶片数量、叶面积、根长、分株和单株鲜重等生长指标,而11:00～15:00遮荫虽抑制叶片数量和子代分株的产生,但促进根的伸长和叶面积的增加,其单株鲜重比对照增加;同时,还可诱导最大净光合速率(Pnmax),超氧化物歧化酶(SOD)、过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)和AOC活性的增加,减少了体内丙二醛(MDA)和超氧阴离子自由基(O2.-)的累积,稳定Rubisco活性,减轻光氧化胁迫。研究表明,全天遮荫不利于凤眼莲的生长,但中午遮荫有利于缓解光氧化伤害,促进其物质累积。     

Antibacterial activity of sugar maple autumn-shed leaf extract: Identification of the active compound

Ethanolic crude extract prepared from autumn-shed leaves of sugar maple (Acer saccharum Marsh.) was recently shown to have antibacterial activity against Pseudomonas cichorii and Xanthomonas campestris pv. vitians, two bacteria causing diseases in lettuce production. In this study, antibacterial activity of sugar maple autumn-shed leaves (SMASL) extract was further investigated. SMASL ethanolic crude extract was fractionated using HPLC system and geraniin was identified as the antibacterial compound by UPLC/Q-Tof-MS system. Geraniin, an ellagitannin, was then purified from SMASL crude extract using a glass chromatographic C18-reversed phase silica gel column (purification Step 1) and a semi-preparative HPLC system equipped with 5 μm XTerra Prep MS C18 column (purification Step 2). Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of purified geraniin (purity of 96%) against P. cichorii and X. campestris pv. vitians were determined. X. campestris pv. vitians (MIC of 0.024 mg ml⁻¹ and MBC of 3.125 mg ml⁻¹) was more sensitive to geraniin than P. cichorii (MIC of 0.781 mg ml⁻¹ and MBC of 6.25 mg ml⁻¹). In the present study, geraniin is reported for the first time as the main antibacterial compound present in SMASL.     

Examining the effects of chronic,lake-wide elevated temperatures on behavioural expression in largemouth bass,Micropterus salmoides

Many behaviours have differential fitness consequences across thermal and ecological contexts, indicating that both ecological shifts and warming temperatures induced by climatic change may alter how organisms behave. However, empirical evidence of temperature-driven behavioural selection in natural systems is lacking. We compared behaviours and behavioural syndromes related to activity, exploration, boldness and aggression in populations of largemouth bass (Micropterus salmoides) from ambient lakes to the those from artificially warmed, power plant cooling lakes to investigate changes in behaviours associated with warmer environments. Activity, exploration, boldness and aggression of juvenile largemouth bass were assessed in laboratory conditions using a novel environment assay and a risky situation assay. We found that activity and exploratory behaviours were higher and decreased through first year ontogeny in populations from heated lakes, whereas these behaviours were lower and showed no relationship through ontogeny in populations from ambient lakes. We attribute these differences to the changes in food source availability in heated lakes associated with temperature-driven ecological effects. Bold and aggressive behaviours tended to differ between populations, as did correlations between behaviours, but did not differ between ambient and heated lakes. The findings of this work identify that large ecological changes associated with warming environments, such as food availability, may drive changes in some aspects of behavioural expression in largemouth bass but that other aspects of behavioural expression may be driven by lake-specific factors not related to warming.     

Comparative metabolomics implicates threitol as a fungal signal supporting colonization of Armillaria luteobubalina on eucalypt roots

Armillaria root rot is a fungal disease that affects a wide range of trees and crops around the world. Despite being a widespread disease, little is known about the plant molecular responses towards the pathogenic fungi at the early phase of their interaction. With recent research highlighting the vital roles of metabolites in plant root–microbe interactions, we sought to explore the presymbiotic metabolite responses of Eucalyptus grandis seedlings towards Armillaria luteobuablina, a necrotrophic pathogen native to Australia. Using a metabolite profiling approach, we have identified threitol as one of the key metabolite responses in E. grandis root tips specific to A. luteobubalina that were not induced by three other species of soil-borne microbes of different lifestyle strategies (a mutualist, a commensalist, and a hemi-biotrophic pathogen). Using isotope labelling, threitol detected in the Armillaria-treated root tips was found to be largely derived from the fungal pathogen. Exogenous application of d- threitol promoted microbial colonization of E. grandis and triggered hormonal responses in root cells. Together, our results support a role of threitol as an important metabolite signal during eucalypt-Armillaria interaction prior to infection thus advancing our mechanistic understanding on the earliest stage of Armillaria disease development. Comparative metabolomics of eucalypt roots interacting with a range of fungal lifestyles identified threitol enrichment as a specific characteristic of Armillaria pathogenesis. Our findings suggest that threitol acts as one of the earliest fungal signals promoting Armillaria colonization of roots.     

Arabinogalactan protein-rich cell walls,paramural deposits and ergastic globules define the hyaline bodies of rhinanthoid Orobanchaceae haustoria

Background and Aims

Parasitic plants obtain nutrients from their hosts through organs called haustoria. The hyaline body is a specialized parenchymatous tissue occupying the central parts of haustoria in many Orobanchaceae species. The structure and functions of hyaline bodies are poorly understood despite their apparent necessity for the proper functioning of haustoria. Reported here is a cell wall-focused immunohistochemical study of the hyaline bodies of three species from the ecologically important clade of rhinanthoid Orobanchaceae.

Methods

Haustoria collected from laboratory-grown and field-collected plants of Rhinanthus minor, Odontites vernus and Melampyrum pratense attached to various hosts were immunolabelled for cell wall matrix glycans and glycoproteins using specific monoclonal antibodies (mAbs).

Key Results

Hyaline body cell wall architecture differed from that of the surrounding parenchyma in all species investigated. Enrichment in arabinogalactan protein (AGP) epitopes labelled with mAbs LM2, JIM8, JIM13, JIM14 and CCRC-M7 was prominent and coincided with reduced labelling of de-esterified homogalacturonan with mAbs JIM5, LM18 and LM19. Furthermore, paramural bodies, intercellular deposits and globular ergastic bodies composed of pectins, xyloglucans, extensins and AGPs were common. In Rhinanthus they were particularly abundant in pairings with legume hosts. Hyaline body cells were not in direct contact with haustorial xylem, which was surrounded by a single layer of paratracheal parenchyma with thickened cell walls abutting the xylem.

Conclusions

The distinctive anatomy and cell wall architecture indicate hyaline body specialization. Altered proportions of AGPs and pectins may affect the mechanical properties of hyaline body cell walls. This and the association with a transfer-like type of paratracheal parenchyma suggest a role in nutrient translocation. Organelle-rich protoplasts and the presence of exceptionally profuse intra- and intercellular wall materials when attached to a nitrogen-fixing host suggest subsequent processing and transient storage of nutrients. AGPs might therefore be implicated in nutrient transfer and metabolism in haustoria.