Deleterious effects of plant cystatins against the banana weevil Cosmopolites sordidus

The general potential of plant cystatins for the development of insect‐resistant transgenic plants still remains to be established given the natural ability of several insects to compensate for the loss of digestive cysteine protease activities. Here we assessed the potential of cystatins for the development of banana lines resistant to the banana weevil Cosmopolites sordidus, a major pest of banana and plantain in Africa. Protease inhibitory assays were conducted with protein and methylcoumarin (MCA) peptide substrates to measure the inhibitory efficiency of different cystatins in vitro, followed by a diet assay with cystatin‐infiltrated banana stem disks to monitor the impact of two plant cystatins, oryzacystatin I (OC‐I, or OsCYS1) and papaya cystatin (CpCYS1), on the overall growth rate of weevil larvae. As observed earlier for other Coleoptera, banana weevils produce a variety of proteases for dietary protein digestion, including in particular Z‐Phe‐Arg‐MCA‐hydrolyzing (cathepsin L–like) and Z‐Arg‐Arg‐MCA‐hydrolyzing (cathepsin B–like) proteases active in mildly acidic conditions. Both enzyme populations were sensitive to the cysteine protease inhibitor E‐64 and to different plant cystatins including OsCYS1. In line with the broad inhibitory effects of cystatins, OsCYS1 and CpCYS1 caused an important growth delay in young larvae developing for 10 days in cystatin‐infiltrated banana stem disks. These promising results, which illustrate the susceptibility of C. sordidus to plant cystatins, are discussed in the light of recent hypotheses suggesting a key role for cathepsin B–like enzymes as a determinant for resistance or susceptibility to plant cystatins in Coleoptera. © 2009 Wiley Periodicals, Inc.     

Determination of puerarin in biological samples and its application to a pharmacokinetic study by flow‐injection chemiluminescence

A simple and sensitive flow injection–chemiluminescence (FI–CL) method has been developed for the determination of puerarin, based on the fact that puerarin can greatly inhibit CL of the luminol–H₂O₂–haemoglobin system. The inhibition of CL intensity was linear to the logarithm of the concentration of puerarin in the range 0.08–10.0 μg/mL (r² = 0.9912). The limit of detection was 0.05 μg/mL (3σ) and the relative standard deviation (RSD) for 1.0 μg/mL (n = 11) of puerarin solution was 1.4%. Coupled with solid‐phase extraction (SPE) as the sample pretreatment, the determination of puerarin in biological samples and a preliminary pharmocokinetic study of puerarin in rats were performed. The recoveries for plasma and urine at three different concentrations were 89.2–110.0% and 91.4–104.8%, respectively. The pharmacokinetics of puerarin in plasma of rat coincides with the two‐compartment open model. The T_1/2α, T_1/2β, CL/F, V_Z/F, AUC_{(0 – t)}, MRT_{(0 – ∞)}, T_max and C_max were 0.77 ± 0.21 h, 7.55 ± 2.64 h, 2.43 ± 1.02 L/kg/h, 11.40 ± 3.45 L/kg, 56.67 ± 10.65 mg/h/L, 5.04 ± 2.78 h, 1.00 ± 0.35 h and 19.70 ± 4.67 μg/mL, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Removal of early fruit branches triggered regulations in senescence,boll attributes and yield of Bt cotton genotypes

Premature senescence, unopened and rotted bolls are the primary constrictions hampering the accomplishment of yield potential in Bt cotton. An experiment was conducted with the objectives to delay senescence, enhance boll opening and reduce the boll rotting. The experiment was conducted at two randomly selected sites of University of Agriculture Faisalabad, Pakistan, during 2015 and repeated in 2016. Each experiment was laid out in randomised complete block design with two by two factorial arrangement structure and replicated five times. Treatments were comprised of R₀ = no fruit removal; R₁ = removal of early fruit branches (REFB) and two genotypes, viz. V₁ = FH‐142 and V₂ = MNH‐886. Genotypes were selected on the basis of abiotic stress tolerance (FH‐142) and susceptibility (MNH‐886). Nodes above cracked bolls, monopodial branches, opened bolls per plant and seed cotton yield were enhanced by 23, 12, 13 and 17%, respectively, under the influence of REFB over no fruit branch removal, while the REFB‐mediated decrease in boll rotting was 27%. Moreover, the response of MNH‐886 was more remarkable than FH‐142 for all studied variables. Conclusively, REFB increased the number of nodes above a cracked boll, monopodial branches, opened bolls per plant and seed cotton yield while decreased rotted bolls per plant in both genotypes.     

Detection of membrane protein–protein interaction in planta based on dual‐intein‐coupled tripartite split‐GFP association

Despite the great interest in identifying protein–protein interactions (PPIs) in biological systems, only a few attempts have been made at large‐scale PPI screening in planta. Unlike biochemical assays, bimolecular fluorescence complementation allows visualization of transient and weak PPIs in vivo at subcellular resolution. However, when the non‐fluorescent fragments are highly expressed, spontaneous and irreversible self‐assembly of the split halves can easily generate false positives. The recently developed tripartite split‐GFP system was shown to be a reliable PPI reporter in mammalian and yeast cells. In this study, we adapted this methodology, in combination with the β‐estradiol‐inducible expression cassette, for the detection of membrane PPIs in planta. Using a transient expression assay by agroinfiltration of Nicotiana benthamiana leaves, we demonstrate the utility of the tripartite split‐GFP association in plant cells and affirm that the tripartite split‐GFP system yields no spurious background signal even with abundant fusion proteins readily accessible to the compartments of interaction. By validating a few of the Arabidopsis PPIs, including the membrane PPIs implicated in phosphate homeostasis, we proved the fidelity of this assay for detection of PPIs in various cellular compartments in planta. Moreover, the technique combining the tripartite split‐GFP association and dual‐intein‐mediated cleavage of polyprotein precursor is feasible in stably transformed Arabidopsis plants. Our results provide a proof‐of‐concept implementation of the tripartite split‐GFP system as a potential tool for membrane PPI screens in planta.     

Ants suppressing plant pathogens: a review

Ant–plant mutualisms are usually regarded as driven by ants defending plants against herbivores in return for plant‐produced food rewards and housing. However, ants may provide additional services. In a review of published studies on ant–pathogen–plant interactions, we investigated whether ants’ extensive hygiene measures, including the use of ant‐produced antibiotics, extend to their host plants and reduce plant pathogen loads. From 30 reported species combinations, we found that the presence of ants lead to reduced pathogen levels in 18 combinations and to increased levels in 6. On average, ants significantly reduced pathogen incidence with 59%. This effect size did not differ significantly from effect sizes reported from meta‐analyses on herbivore protection. Thus, pathogen and herbivore protection could be of equal importance in ant–plant mutualisms. Considering the abundance of these interactions, ecological impacts are potentially high. Furthermore, awareness of this service may stimulate the development of new measures to control plant diseases in agriculture. It should be noted, though, that studies were biased toward tropical ant–plant symbioses and that the literature in the field is limited at present. Future research on plant pathogens is needed to enhance our understanding of ant–plant mutualisms and their evolution.     

Above‐ground biomass distribution among species during early old‐field succession

Abstract. The interdependence of species richness and plant biomass has widely been accepted as a general biodiversity rule. However, there is no information about how relationships are established during colonization and how total biomass is distributed among plants. The main objective of this study was to determine the role of several factors which we have hypothetized as affecting biomass distribution among species in an early old‐field community. To test this hypothesis, we conducted an experiment in a deeply ploughed Agropyro‐Rumicion crispi community in the Basque Country (Spain). Light, water and soil nitrogen content were factorially manipulated resulting in eight experimental treatments. We also examined several site features, which could potentially influence final biomass distribution: seed bank composition, soil physico‐chemical heterogeneity and cover and density of the dominant plant species in the community. A partition hypothesis testing approach using Redundancy Analysis was conducted to determine the fraction of biomass distribution variability assigned to each treatment and site feature. The most important species, in terms of biomass, were Agropyron repens, Sinapis arvensis, Arrhenatherum bulbosum and Picris echioides. As a general conclusion, biomass distribution among species during early secondary succession primarily depends on light availability and nitrogen supply. Several soil variables, such as magnesium, calcium and clay contents, also explain a relevant fraction of the biomass distribution among plant species. On the contrary, we found no effect of seed bank composition on biomass distribution. Finally, the total species number and cover of dominant species such as Sinapis arvensis, may determine final biomass distribution.     

The Botrytis cinerea early secretome

The extracellular proteome, or secretome, of phytopathogenic fungi is presumed to be a key element of their infection strategy. Especially interesting constituents of this set are those proteins secreted at the beginning of the infection, during the germination of conidia on the plant surfaces or wounds, since they may play essential roles in the establishment of a successful infection. We have germinated Botrytis cinerea conidia in conditions that resemble the plant environment, a synthetic medium enriched with low molecular weight plant compounds, and we have collected the proteins secreted during the first 16 h by a double precipitation protocol. 2‐D electrophoresis of the precipitated secretome showed a spot pattern similar for all conditions evaluated and for the control medium without plant extract. The proteins in 16 of these spots were identified by PMF and corresponded to 11 different polypeptides. Alternative determination of secretome composition by LC‐MS/MS of tryptic fragments rendered a much larger number, 105 proteins, which included all previously identified by PMF. All proteins were functionally classified according to their putative function in the infection process. Key features of the early secretome include a large number of proteases, the abundance of proteins involved in the degradation of plant defensive barriers, and plenty of proteins with unknown function.     

A vegetation analysis of the pastoral landscapes of upland Wales,UK

Abstract. The upland moorlands of Wales are situated on the oceanic fringe of western Europe, and have experienced a long history of pastoral management. Recent vegetation data are analysed to assess the relative contribution of abiotic and anthropogenic factors to variation in habitat composition among the major upland ranges of this region. From a numerical analysis of plant community cover data, recorded from 65 sites covering 260 000 ha, a six‐cluster site classification emerged with striking biogeographical coherence. Direct gradient analysis and variance partitioning revealed strong correlation between vegetation composition and spatially‐structured climatic gradients, in particular temperature, rainfall and oceanicity; differences in bedrock geology appear to have a lesser role. The analysis also indicates a close correlation between habitat variation and anthropogenic parameters, especially grazing intensity, burning frequency, and sulphur and nitrogen deposition levels. At this regional scale, anthropogenic impacts appear to have accentuated, rather than obscured, vegetation patterns which are primarily determined by climate and other abiotic variables. The findings have considerable relevance for conservation planning and also for predictive studies on the consequences of climatic change for the biota of the uplands of southern Britain.