Lipoxygenases regulate digestive enzyme inhibitor activities in developing seeds of field-grown soybean against the southern green stink bug (Nezara viridula).

Soybean (Glycine max ) is the world's most widely grown seed legume. One of the most important pests that decrease seed quality and reduce yield of soybean crops is the southern green stink bug (Nezara viridula ). Insect damage triggers accumulation of defensive compounds such as protease inhibitors (PIs), isoflavonoids and reactive oxygen species, which are regulated by the lipoxygenase (LOX)-regulated jasmonic acid (JA) to stop insect feeding. This study identified and characterised the role of LOX isoforms in the modulation of chemical defences in seeds of field-grown soybean that decreased digestive enzyme activities of N. viridula after insect attack. Stink bugs attack increased LOX 1 and LOX 2 expression, and activities of LOX 1 and LOX 3 isoenzymes in developing soybean seeds. In addition, stink bug damage and methyl jasmonate application induced expression and activity of both cysteine PIs and trypsin PIs in developing soybean seeds, suggesting that herbivory induced JA in soybean seeds. High PI activity levels in attacked seeds decreased cysteine proteases and α-amylases activities in the gut of stink bugs that fed on field-grown soybean. We demonstrated that LOX isoforms of seeds are concomitantly induced with JA-regulated PIs by stink bugs attack, and these PIs inhibit the activity of insect digestive enzymes. To our knowledge, this is the first study to investigate the participation of LOX in modulating JA-regulated defences against stink bugs in seeds of field-grown soybean, and our results suggest that soybean PIs may inhibit α-amylase activity in the gut of N. viridula .

Feeding on soybean crops changed gut bacteria diversity of the southern green stinkbug (Nezara viridula) and reduced negative effects of some associated bacteria.

BACKGROUND: The southern green stinkbug (Nezara viridula) is a mayor pest of soybean. However, the mechanism underlying stinkbug resistance to soybean defenses is yet ignored. Although gut bacteria could play an essential role in tolerating plant defenses, most studies testing questions related to insect-plant-bacteria interactions have been performed in laboratory condition. Here we performed experiments in laboratory and field conditions with N. viridula and its gut bacteria, studying gut lipid peroxidaxion levels and cysteine activity in infected and unifected nymphs, testing the hypothesis that feeding on field-grown soybean decreases bacterial abundance in stinkbugs. RESULTS: Gut bacterial abundance and infection ratio were higher in N. viridula adults reared in laboratory than in those collected from soybean crops, suggesting that stinkbugs in field conditions may modulate gut bacterial colonization. Manipulating gut microbiota by infecting stinkbugs with Yokenella sp. showed that these bacteria abundance decreased in field conditions, and negatively affected stinkbugs performance and were more aggressive in laboratory rearing than in field conditions. Infected nymphs that fed on soybean pods had lower mortality, higher mass and shorter development period than those reared in the laboratory, and suggested that field conditions helped nymphs to recover from Yokenella sp. infection, despite of increased lipid peroxidation and decreased cysteine proteases activity in nymphs' guts. CONCLUSIONS: Our results demonstrated that feeding on field-grown soybean reduced bacterial abundance and infection in guts of N. viridula and highlighted the importance to test functional activities or pathogenicity of microbes under realistic field conditions prior to establish conclusions on three trophic interactions. © 2022 Society of Chemical Industry.

Proteases inhibitors-insensitive cysteine proteases allow Nezara viridula to feed on growing seeds of field-grown soybean.

The southern green stink bug, Nezara viridula is one of the primary soybean pests and causes significant economic losses around the world. In spite of the high proteases inhibitor (PI) levels, N. viridula can feed on developing seeds of field-grown soybean and reduce crop yields. Although the PI-induced responses have been extensively investigated in many pest insects, there is lack of knowledge about the mechanisms that stink bugs employ to withstand cysteine PIs of soybean seeds. This study demonstrated that feeding on developing seeds of field-grown soybean inhibited total proteases activity of N. viridula, as result of inhibition of cathepsin B-like activity in the gut. In addition, from the 30 digestive cathepsins recognized in this study, 6 were identified as cathepsin B-like. Stink bugs that fed on growing seeds of field-grown soybean had similar gut pH to those reared in the laboratory, and both cathepsin B- and L-like had an optima pH of 6.5. Therefore, using specific proteases inhibitors we found that the main proteolytic activity in the gut is from cysteine proteases when N. viridula feeds on soybean crops. Since cathepsin L-like activity was not inhibited by soybean PIs, our results suggested that N. viridula relays on cathepsin L-like to feed on soybean. To our knowledge no study before has shown the impact of seed PIs of field-grown soybean on digestive proteases (cathepsin B- and L-like) of N. viridula. This study suggests that the activity of PI-insensitive cathepsins L-like in the gut would be part of an adaptive strategy to feed on developing soybean seeds. In agreement, the expansions of cathepsin L-like complement observed in pentatomids could confer to the insects a higher versatility to counteract the effects of different PIs.

Evaluating the Impact of Post-Emergence Weed Control in Honeybee Colonies Located in Different Agricultural Surroundings.

The honeybee Apis mellifera is exposed to agricultural intensification, which leads to an improved reliance upon pesticide use and the reduction of floral diversity. In the present study, we assess the changes in the colony activity and the expression profile of genes involved in xenobiotic detoxification in larvae and adult honeybees from three apiaries located in agricultural environments that differ in their proportion of the crop/wild flora. We evaluated these variables before and after the administration of a mixture of three herbicides during the summer season. The expression of several cytochrome P450 monooxygenases decreased significantly in larvae after post-emergence weed control and showed significant differences between apiaries in the case of honeybee workers. Principal component analysis (PCA) revealed that colonies located in the plot near to a wetland area exhibited a different relative gene expression profile after herbicide application compared with the other plots. Moreover, we found significant positive correlations between pollen collection and the pesticide detoxification genes that discriminated between plots in the PCA. Our results suggest that nutrition may modify herbicide impact on honeybees and that larvae are more harmed than adults in agroecosystems, a factor that will alter the colonies' population growth at the end of the blooming period.

Role of reactive oxygen species and isoflavonoids in soybean resistance to the attack of the southern green stink bug.

Southern green stink bugs (Nezara viridula L.) are one of the major pests in many soybean producing areas. They cause a decrease in yield and affect seed quality by reducing viability and vigor. Alterations have been reported in the oxidative response and in the secondary metabolites in different plant species due to insect damage. However, there is little information available on soybean-stink bug interactions. In this study we compare the response of undamaged and damaged seeds by Nezara viridula in two soybean cultivars, IAC-100 (resistant) and Davis (susceptible), grown under greenhouse conditions. Pod hardness, H2O2 generation, enzyme activities in guaiacol peroxidase (GPOX), catalase (CAT) and superoxide dismutase (SOD) as well as lipoxygenase expression and isoflavonoid production were quantified. Our results showed a greater resistance of IAC-100 to pod penetration, a decrease in peroxide content after stink bug attack, and higher GPOX, CAT and SOD activities in seeds due to the genotype and to the genotype-interaction with the herbivory treatment. Induction of LOX expression in both cultivars and higher production of isoflavonoids in IAC-100 were also detected. It was then concluded that the herbivory stink bug induces pathways related to oxidative stress and to the secondary metabolites in developing seeds of soybean and that differences between cultivars hold promise for a plant breeding program.

Glyphosate affects the larval development of honey bees depending on the susceptibility of colonies.

As the main agricultural insect pollinator, the honey bee (Apis mellifera) is exposed to a number of agrochemicals, including glyphosate (GLY), the most widely used herbicide. Actually, GLY has been detected in honey and bee pollen baskets. However, its impact on the honey bee brood is poorly explored. Therefore, we assessed the effects of GLY on larval development under chronic exposure during in vitro rearing. Even though this procedure does not account for social compensatory mechanisms such as brood care by adult workers, it allows us to control the herbicide dose, homogenize nutrition and minimize environmental stress. Our results show that brood fed with food containing GLY traces (1.25-5.0 mg per litre of food) had a higher proportion of larvae with delayed moulting and reduced weight. Our assessment also indicates a non-monotonic dose-response and variability in the effects among colonies. Differences in genetic diversity could explain the variation in susceptibility to GLY. Accordingly, the transcription of immune/detoxifying genes in the guts of larvae exposed to GLY was variably regulated among the colonies studied. Consequently, under laboratory conditions, the response of honey bees to GLY indicates that it is a stressor that affects larval development depending on individual and colony susceptibility.

An Early Sensitive Period Induces Long-Lasting Plasticity in the Honeybee Nervous System.

The effect of early experiences on the brain during a sensitive period exerts a long-lasting influence on the mature individual. Despite behavioral and neural plasticity caused by early experiences having been reported in the honeybee Apis mellifera, the presence of a sensitive period in which associative experiences lead to pronounced modifications in the adult nervous system is still unclear. Laboratory-reared bees were fed with scented food within specific temporal windows and were assessed for memory retention, in the regulation of gene expression related to the synaptic formation and in the olfactory perception of their antennae at 17 days of age. Bees were able to retain a food-odor association acquired 5-8 days after emergence, but not before, and showed better retention than those exposed to an odor at 9-12 days. In the brain, the odor-rewarded experiences that occurred at 5-8 days of age boosted the expression levels of the cell adhesion proteins neurexin 1 (Nrx1) and neuroligin 2 (Nlg2) involved in synaptic strength. At the antennae, the experiences increased the electrical response to a novel odor but not to the one experienced. Therefore, a sensitive period that induces long-lasting behavioral, functional and structural changes is found in adult honeybees.

Solar UV-B radiation and ethylene play a key role in modulating effective defenses against Anticarsia gemmatalis larvae in field-grown soybean.

Solar UV-B radiation has been reported to enhance plant defenses against herbivore insects in many species. However, the mechanism and traits involved in the UV-B mediated increment of plant resistance are unknown in crops species, such as soybean. Here, we studied defense-related responses in undamaged and Anticarsia gemmatalis larvae-damaged leaves of two soybean cultivars grown under attenuated or full solar UV-B radiation. We determined changes in jasmonates, ethylene (ET), salicylic acid, trypsin protease inhibitor activity, flavonoids, and mRNA expression of genes related with defenses. ET emission induced by Anticarsia gemmatalis damage was synergistically increased in plants grown under solar UV-B radiation and was positively correlated with malonyl genistin concentration, trypsin proteinase inhibitor activity and expression of IFS2, and the pathogenesis protein PR2, while was negatively correlated with leaf consumption. The precursor of ET, aminocyclopropane-carboxylic acid, applied exogenously to soybean was sufficient to strongly induce leaf isoflavonoids. Our results showed that in field-grown soybean isoflavonoids were regulated by both herbivory and solar UV-B inducible ET, whereas flavonols were regulated by solar UV-B radiation only and not by herbivory or ET. Our study suggests that, although ET can modulate UV-B-mediated priming of inducible plant defenses, some plant defenses, such as isoflavonoids, are regulated by ET alone.

Early perception of stink bug damage in developing seeds of field-grown soybean induces chemical defences and reduces bug attack.

BACKGROUND: Southern green stink bugs (Nezara viridula L.) invade field-grown soybean crops, where they feed on developing seeds and inject phytotoxic saliva, which causes yield reduction. Although leaf responses to herbivory are well studied, no information is available about the regulation of defences in seeds. RESULTS: This study demonstrated that mitogen-activated protein kinases MPK3, MPK4 and MPK6 are expressed and activated in developing seeds of field-grown soybean and regulate a defensive response after stink bug damage. Although 10-20 min after stink bug feeding on seeds induced the expression of MPK3, MPK6 and MPK4, only MPK6 was phosphorylated after damage. Herbivory induced an early peak of jasmonic acid (JA) accumulation and ethylene (ET) emission after 3 h in developing seeds, whereas salicylic acid (SA) was also induced early, and at increasing levels up to 72 h after damage. Damaged seeds upregulated defensive genes typically modulated by JA/ET or SA, which in turn reduced the activity of digestive enzymes in the gut of stink bugs. Induced seeds were less preferred by stink bugs. CONCLUSION: This study shows that stink bug damage induces seed defences, which is perceived early by MPKs that may activate defence metabolic pathways in developing seeds of field-grown soybean. © 2015 Society of Chemical Industry.

A Japanese plum α-l-arabinofuranosidase/ß-D-xylosidase gene is developmentally regulated by alternative splicing.

A full-length cDNA clone named PsARF/XYL was obtained from Prunus salicina Lindl., and determined to encode a putative α-l-arabinofuranosidase/ß-d-xylosidase belonging to glycoside hydrolase (GH, EC 3.2.1.-) family 3. Two related PsARF/XYL cDNAs were amplified, one from a fully-spliced transcript (PsARF/XYLa) and another one from an intron-retained transcript (PsARF/XYLb). The protein deduced from PsARF/XYLb is a truncated peptide at C-terminus that conserves the active-site amino acid sequence. High levels of PsARF/XYLa and PsARF/XYLb transcripts are detectable in several plant tissues. PsARF/XYLb transcripts accumulate progressively during the phase of exponential fruit growth but they become barely noticeable during on-tree ripening, or after a 6-h exposure of preclimacteric full-size plums to ethylene. In contrast, PsARF/XYLa is expressed throughout fruit development, and transcript accumulation parallels the climacteric rise in ethylene production during ripening. PsARF/XYLa expression is strongly induced in preclimacteric full-size plums after a 6-h treatment with physiologically active concentrations of ethylene. These findings suggest that PsARF/XYL gene is post-transcriptionally regulated by alternative splicing during development and that ethylene may be involved in this regulation. The isolation of a partial cDNA clone, PsARF1, is also reported. It encodes a putative cell-wall α-l-arabinofuranosidase, and its transcription is rapidly inhibited by ethylene in mature green plums.

Exposure assessment to glyphosate of two species of annelids.

Adult mortality, biomass, fecundity and viability of cocoons were studied in Eisenia fetida and Octolasion tyrtaeum, in response to glyphosate exposure in soil. Exposure tests were carried out following USEPA procedure, with five concentrations of glyphosate in soil and a control. O. tyrtaeum was more sensitive to the highest concentration of glyphosate (50,000 mg kg(-1)), with 100 % mortality by day 7 of exposure, compared with 71 % for E. fetida. Although biomass of O. tyrtaeum was significantly different between the control and 5,000 mg kg(-1) dose at day 14, E. fetida was not affected at that concentration, and only showed a significant weight loss after 7 days of exposure to 50,000 mg kg(-1). Adverse effects upon adult fecundity and cocoon viability were observed at glyphosate concentrations of 5,000 mg kg(-1) and above. Adverse effects were observed at concentrations that greatly exceeded the recommended field application rates of glyphosate.

Uptake of chromium by Salvinia minima: effect on plant growth, leaf respiration and carbohydrate metabolism.

Metabolic responses to chromium (Cr) exposure and metal uptake were investigated using Salvinia minima plants. Cr treatment reduced the dry weight of floating and submerged leaves, while photosynthetic pigments were not affected. Measurements of respiratory oxygen uptake with and without inhibitors (KCN and SHAM) demonstrated that total respiration, alternative oxidase capacity and residual respiration were higher in Cr-treated than in Cr-untreated leaves, but the highest values were observed in floating leaves. Cr affected the soluble sugar content. Sucrose concentration was, in general, higher in Cr-treated than in Cr-untreated leaves, while the glucose concentration showed an inverse pattern. Cr also affected soluble acid invertase activity, but affectation trend was different between both leaves. Highest values of invertase activity were observed in Cr-treated floating leaves. According to our data soluble acid invertase and sucrose seem to be related to alternative oxidase capacity and residual respiration in floating and submerged leaves exposed to Cr. Thereby, this study constitutes an important contribution to understand metabolic relationships between mitochondrial respiration, alternative respiratory pathway and soluble carbohydrates in plants exposed to heavy metals.

Differential expression of alpha-l-arabinofuranosidase and alpha-l-arabinofuranosidase/beta-d-xylosidase genes during peach growth and ripening.

Arabinose is the major neutral sugar in peach (Prunus persica (L.) Batsch) cell walls and substantial changes in arabinose content take place not only during peach melting, when a rapid-softening-related depolymerizing activity may be expected, but also at the onset of peach ripening. A full-length cDNA clone sequence referred to as PpARF1 (GenBank accession no. DQ486870) was obtained and determined by bioinformatics' analysis to be a peach alpha-l-arabinofuranosidase homologue. The deduced PpARF1 translation product is 677 amino acids in length while the mature protein has a predicted molecular mass of 71.6 kD and a theoretical pI of 4.94. Semi-quantitative RT-PCR reactions were conducted to evaluate the expression of both PpARF1 and PpARF/XYL (GenBank accession no. AB264280), the latter encoding a putative bifunctional protein displaying both alpha-l-arabinofuranosidase and beta-d-xylosidase activities. In peach fruit, the PpARF1 gene expression was detected at every developmental stage with a maximum during S2 (lag phase of development) and a subsequent decrease towards S4 (maximal fruit size). In contrast, PpARF/XYL transcript levels were relatively high at the end of S1 (fruit set) and at S3-E (beginning of the cell expansion). Substantial increases in PpARF1 mRNA levels were found at the beginning and end of the climacteric rise and also in melting fruit. In contrast, PpARF/XYL transcripts reached a maximum when fruit firmness was 22-26 N, with a slight decline during the melting stage. PpARF/XYL and PpARF1 were expressed differently in three fruit tissue types as well as in other plant tissues. Ethylene is regarded as the main regulator of peach ripening and the accumulation of PpARF/XYL and PpARF1 transcripts is coincident with the autocatalytic ethylene production during ripening. On the hand, other factors may also play a role in PpARF1 and PpARF/XYL expression, since transcripts accumulate at different developmental times and organs even when ethylene biosynthesis is barely detectable.

Chloroplast fructose-1,6-bisphosphatase: structure and function.

Redox regulation of photosynthetic enzymes has been a preferred research topic in recent years. In this area chloroplast fructose-1,6-bisphosphatase is probably the most extensively studied target enzyme of the CO(2) assimilation pathway. This review analyzes the structure, biosynthesis, phylogeny, action mechanism, regulation and kinetics of fructose-1,6-bisphosphatase in the light of recent findings on structure-function relationship, and from a molecular biology viewpoint.