Biotic Defense Research Articles

A nucleoside diphosphate kinase gene OsNDPK4 is involved in root development and defense responses in rice (Oryza sativa L.)

Dysfunctional mutation of OsNDPK4 resulted in severe defects in root development of rice. However, the resistance of Osndpk4 against bacterial blight was significantly enhanced. Nucleoside diphosphate kinases (NDPKs) are an evolutionarily conserved family of important enzymes balancing the energy currency nucleoside triphosphates by catalyzing the transfer of their phosphate groups. The aim of this study was to elucidate the function of OsNDPK4 in rice. A dysfunctional rice mutant was employed to characterize the function of OsNDPK4. Its expression and subcellular localization were examined. The transcriptomic change in roots of Osndpk4 was analyzed by RNA-seq. The rice mutant Osndpk4 showed severe defects in root development from the early seedling stage. Further analysis revealed that meristematic activity and cell elongation were significantly inhibited in primary roots of Osndpk4, together with reduced accumulation of reactive oxygen species (ROS). Map-based cloning identified that the mutation occurred in the OsNDPK4 gene. OsNDPK4 was found to be expressed in a variety of tissues throughout the plant and OsNDPK4 was located in the cytosol. Osndpk4 showed enhanced resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) and up-regulation of pathogenesis-related marker genes. In addition, transcriptomic analysis showed that OsNDPK4 was significantly associated with a number of biological processes, including translation, protein modification, metabolism, biotic stress response, etc. Detailed analysis revealed that the dysfunction of OsNDPK4 might reorchestrate energy homeostasis and hormone metabolism and signalling, resulting in repression of translation, DNA replication and cell cycle progression, and priming of biotic stress defense. Our results demonstrate that OsNDPK4 plays important roles in energy homeostasis, development process, and defense responses in rice.

Relative contribution of ecological and biological attributes in the fine-grain structure of ant-plant networks.

BackgroundEcological communities of interacting species analyzed as complex networks have shown that species dependence on their counterparts is more complex than expected at random. As for other potentially mutualistic interactions, ant-plant networks mediated by extrafloral nectar show a nested (asymmetric) structure with a core of generalist species dominating the interaction pattern. Proposed factors structuring ecological networks include encounter probability (e.g., species abundances and habitat heterogeneity), behavior, phylogeny, and body size. While the importance of underlying factors that influence the structure of ant-plant networks have been separately explored, the simultaneous contribution of several biological and ecological attributes inherent to the species, guild or habitat level has not been addressed.MethodsFor a tropical seasonal site we recorded (in 48 censuses) the frequency of pairwise ant-plant interactions mediated by extrafloral nectaries (EFN) on different habitats and studied the resultant network structure. We addressed for the first time the role of mechanistic versus neutral determinants at the ‘fine-grain’ structure (pairwise interactions) of ant-plant networks. We explore the simultaneous contribution of several attributes of plant and ant species (i.e., EFN abundance and distribution, ant head length, behavioral dominance and invasive status), and habitat attributes (i.e., vegetation structure) in prevailing interactions as well as in overall network topology (community).ResultsOur studied network was highly-nested and non-modular, with core species having high species strengths (higher strength values for ants than plants) and low specialization. Plants had higher dependences on ants than vice versa. We found that habitat heterogeneity in vegetation structure (open vs. shaded habitats) was the main factor explaining network and fine-grain structure, with no evidence of neutral (abundance) effects.DiscussionCore ant species are relevant to most plants species at the network showing adaptations to nectar consumption and deterrent behavior. Thus larger ants interact with more plant species which, together with higher dependence of plants on ants, suggests potential biotic defense at a community scale. In our study site, heterogeneity in the ant-plant interactions among habitats is so prevalent that it emerges at community-level structural properties. High frequency of morphologically diverse and temporarily-active EFNs in all habitats suggests the relevance and seasonality of plant biotic defense provided by ants. The robust survey of ecological interactions and their biological/ecological correlates that we addressed provides insight of the interplay between adaptive-value traits and neutral effects in ecological networks.

MiR159 Represses a Constitutive Pathogen Defense Response in Tobacco.

MicroR159 (miR159) regulation of GAMYB expression is highly conserved in terrestrial plants; however, its functional role remains poorly understood. In Arabidopsis (Arabidopsis thaliana), although GAMYB-like genes are constitutively transcribed during vegetative growth, their effects are suppressed by strong and constitutive silencing by miR159. GAMYB expression occurs only if miR159 function is inhibited, which results in detrimental pleiotropic defects, questioning the purpose of the miR159-GAMYB pathway. Here, miR159 function was inhibited in tobacco (Nicotiana tabacum) and rice (Oryza sativa) using miRNA MIM159 technology. Similar to observations in Arabidopsis, inhibition of miR159 in tobacco and rice resulted in pleiotropic defects including stunted growth, implying functional conservation of the miR159-GAMYB pathway among angiosperms. In MIM159 tobacco, transcriptome profiling revealed that genes associated with defense and programmed cell death were strongly activated, including a suite of 22 PATHOGENESIS-RELATED PROTEIN (PR) genes that were 100- to 1,000-fold upregulated. Constitutive expression of a miR159-resistant GAMYB transgene in tobacco resulted in phenotypes similar to that of MIM159 tobacco and activated PR gene expression, verifying the dependence of the above-mentioned changes on GAMYB expression. Consistent with the broad defense response, MIM159 tobacco appeared immune to Phytophthora infection. These findings suggest that the tobacco miR159-GAMYB pathway functions in the biotic defense response, which becomes activated upon miR159 inhibition. However, PR gene expression was not upregulated in Arabidopsis or rice when miR159 was inhibited, suggesting that miR159-GAMYB pathway functional differences exist between species, or factors in addition to miR159 inhibition are required in Arabidopsis and rice to activate this broad defense response.

Comparative\u2002transcriptome\u2002analysis in Triticum aestivum infecting wheat dwarf virus reveals the effects of viral infection on phytohormone and photosynthesis metabolism pathways

Wheat dwarf virus (WDV), a mastrevirus transmitted by the leafhopper Psammotettix alienus, causes a severe disease in cereal crops. Typical symptoms of wheat plants infected by WDV are yellowing and severe dwarfing. In this present study, RNA-Seq was used to perform gene expression analysis in wheat plants in response to WDV infection. Comparative transcriptome analysis indicated that a total of 1042 differentially expressed genes (DEGs) were identified in the comparison between mock and WDV-inoculated wheat plants. Genomes ontology (GO) annotation revealed a number of DEGs associated with different biological processes, such as phytohormone metabolism, photosynthesis, DNA metabolic process, response to biotic stimulus and defense response. Among these, DEGs involved in phytohormone and photosynthesis metabolism and response pathways were further enriched and analyzed, which indicated that hormone biosynthesis, signaling and chloroplast photosynthesis-related genes might play an important role in symptom development after WDV infection. These results illustrate the dynamic nature of the wheat-WDV interaction at the transcriptome level and confirm that symptom development is a complex process, providing a solid foundation to elucidate the pathogenesis of WDV.

Identification of stress defensive proteins in common wheat-Thinopyron intermedium translocation line YW642 developing grains via comparative proteome analysis.

Thinopyrum intermedium (2n = 6x = 42, E1E1E2E2XX) serves as an important gene source of desirable traits for genetic improvement of wheat cultivars resistant to stresses. This study used the comparative proteomic approach to identify stress defense related proteins in the developing grains of common wheat (Zhongmai 8601)-Thinopyron intermedium 7XL/7DS translocation line YW642 and to explore their potential values for improving wheat stress resistance. Two-dimensional electrophoresis identified 124 differentially accumulated protein spots representing 100 unique proteins, which mainly participated in stress defense, energy metabolism, protein metabolism and folding and storage protein synthesis. Among these, 16 were unique and 35 were upregulated in YW642. The upregulated DAPs were mainly involved in biotic and abiotic stress defense. Further cis-elements analysis of these stress-related DAP genes revealed that phytohormone responsive elements such as ABREs, G-box, CGTCA-motif and TGACG-motif, and environment responsive element As-1 were particularly abundant, which could play important roles in response to various stressors. Transcription expression analysis by RNA-seq and qRT-PCR demonstrated a large part of the stress-related DAP genes showed an upregulated expression in the early-to-middle stages of grain development. Our results proved that Thinopyron intermedium contains abundant stress responsive proteins that have potential values for the genetic improvement of wheat stress resistance.

Vitamin C in Plants: Novel Concepts, New Perspectives, and Outstanding Issues.

Significance: The concept that vitamin C (l-ascorbic acid) is at the heart of the peroxide processing and redox signaling hub in plants is well established, but our knowledge of the precise mechanisms involved remains patchy at best. Recent Advances: Ascorbate participates in the multifaceted signaling pathways initiated by both reactive oxygen species (ROS) and reactive nitrogen species. Crucially, the apoplastic ascorbate/dehydroascorbate (DHA) ratio that is regulated by ascorbate oxidase (AO) sculpts the apoplastic ROS (apoROS) signal that controls polarized cell growth, biotic and abiotic defences, and cell to cell signaling, as well as exerting control over the light-dependent regulation of photosynthesis. Critical Issues: Here we re-evaluate the roles of ascorbate in photosynthesis and other processes, addressing the question of how much we really know about the regulation of ascorbate homeostasis and its functions in plants, or how AO is regulated to modulate apoROS signals. Future Directions: The role of microRNAs in the regulation of AO activity in relation to stress perception and signaling must be resolved. Similarly, the molecular characterization of ascorbate transporters and mechanistic links between photosynthetic and respiratory electron transport and ascorbate synthesis/homeostasis are a prerequisite to understanding ascorbate homeostasis and function. Similarly, there is little in vivo evidence for ascorbate functions as an enzyme cofactor.

Direct comparison of Arabidopsis gene expression reveals different responses to melatonin versus auxin

BackgroundMelatonin (N-acetyl-5-methoxytryptamine) in plants, regulates shoot and root growth and alleviates environmental stresses. Melatonin and the phyto-hormone auxin are tryptophan-derived compounds. However, it largely remains controversial as to whether melatonin and auxin act through similar or overlapping signalling and regulatory pathways.ResultsHere, we have used a promoter-activation study to demonstrate that, unlike auxin (1-naphthalene acetic acid, NAA), melatonin neither induces Direct repeat 5 DR5 expression in Arabidopsis thaliana roots under normal growth conditions nor suppresses the induction of Alternative oxidase 1a AOX1a in leaves upon Antimycin A treatment, both of which are the hallmarks of auxin action. Additionally, comparative global transcriptome analysis conducted on Arabidopsis treated with melatonin or NAA revealed differences in the number and types of differentially expressed genes. Auxin (4.5 μM) altered the expression of a diverse and large number of genes whereas melatonin at 5 μM had no significant effect but melatonin at 100 μM had a modest effect on transcriptome compared to solvent-treated control. Interestingly, the prominent category of genes differentially expressed upon exposure to melatonin trended towards biotic stress defence pathways while downregulation of key genes related to photosynthesis was observed.ConclusionTogether these findings indicate that though they are both indolic compounds, melatonin and auxin act through different pathways to alter gene expression in Arabidopsis thaliana. Furthermore, it appears that effects of melatonin enable Arabidopsis thaliana to prioritize biotic stress defence signalling rather than growth. These findings clear the current confusion in the literature regarding the relationship of melatonin and auxin and also have greater implications of utilizing melatonin for improved plant protection.

The geographical and seasonal mosaic in a plant-herbivore interaction: patterns of defences and herbivory by a specialist and a non-specialist

In order to evaluate the geographic mosaic theory of coevolution, it is crucial to investigate geographical variation on the outcome of ecological interactions and the functional traits which dictate these outcomes. Plant populations are attacked by specialist and non-specialist herbivores and may have different types of chemical and biotic defences. We investigated geographical and seasonal variation in the interaction between the plant Crotalaria pallida and its two major herbivores (the specialist Utetheisa ornatrix and the non-specialist Etiella zinckenella). We first showed that attack by the two herbivores and a chemical and a biotic defence vary greatly in time and space. Second, we performed a common garden experiment that revealed genetic variation among populations in herbivore resistance and a chemical defence, but no genetic variation in a biotic defence. Third, we sampled 20 populations on a much larger geographical scale and showed great variation in attack rates by the two herbivores and a chemical defence. Finally, we showed that herbivory is not correlated with a chemical defence in the 20 field populations. Our study shows that to understand the evolution of ecological interactions it is crucial to investigate how the outcome of the interaction and the important species traits vary geographically and seasonally.

Natural history and ecology of foraging of the Camponotus crassus Mayr, 1862 (Hymenoptera: Formicidae)

ABSTRACTAnts are abundant in terrestrial ecosystems, especially in the Brazilian Cerrado, where they can play several roles at different levels of the food chain, including protection of plants against herbivores. Although there are many studies that evaluate the ant–plant interaction in the Cerrado, little is known about the natural history of most species of ants. Camponotus crassus Mayr, 1862, for example, is considered one of the main agents of plant biotic defence in Cerrado. But there are no studies specifically focused on this species, which hinders the understanding of how arthropod–plant interactions are structured in Cerrado. Here, we describe the natural history and ecology of the foraging of the C. crassus. We conducted the study from January 2013 to December 2014 in 10 quadrants of 40 m2 to measure: the abundance, density and distribution of nests, location of the nests, the internal structure of the nests, the daily foraging of workers out of the nest, the food items they collect and the existence of territoriality and dominance of the workers on the soil and vegetation. We found 18 nests, 13 in the soil and 5 in hollow trunks on the ground with variable internal structures. The distribution of nests is aggregate, with density of 0.045 nests/m2 and average distance of 3.73 m between nests. The foraging activity occurs on the daytime during the rainy and dry season. Extrafloral nectar and honeydew were the resources most collected, comprising 83.33% of the resources in the rainy period and 30% in the dry period. Camponotus crassus is a dominant species, especially on vegetation, although it also forages on the soil. This is the first study to evaluate in detail the natural history and foraging ecology of C. crassus, a diurnal, aggressive and territorial ant that mainly forage climbing onto the plants.

Extrafloral nectary-bearing plant Mallotus japonicus uses different types of extrafloral nectaries to establish effective defense by ants

Extrafloral nectary (EFN)-bearing plants attract ants to gain protection against herbivores. Some EFN-bearing plants possess different types of EFNs, which might have different effects on ants on the plants. Mallotus japonicus (Thunb.) Muell. Arg. (Euphorbiaceae) bears two types of EFNs, including a pair of large EFNs at the leaf base and many small EFNs along the leaf edge. This study aimed to determine the different roles of the two types of EFNs in biotic defense by ants. A field experiment was conducted to investigate the effect of leaf damage on EFN production and on the distribution pattern of ants. After leaf damage, the number of leaf edge EFNs increased in the leaves first-produced. The number of ants on the leaves also increased, and the foraging area of ants extended from the leaf base to the leaf tip. An EFN-covering field experiment revealed that leaf edge EFNs had a greater effect than leaf base EFNs on ant dispersal on leaves. The extended foraging area of ants resulted in an increase of encounter or attack rate against an experimentally placed herbivore, Spodoptera litura. These results suggest that M. japonicus plants control the foraging area of ants on their leaves using different types of EFNs in response to leaf damage, thus achieving a very effective biotic defense against herbivores by ants.

Turnip Mosaic Virus Components Are Released into the Extracellular Space by Vesicles in Infected Leaves.

Turnip mosaic virus (TuMV) reorganizes the endomembrane system of the infected cell to generate endoplasmic-reticulum-derived motile vesicles containing viral replication complexes. The membrane-associated viral protein 6K2 plays a key role in the formation of these vesicles. Using confocal microscopy, we observed that this viral protein, a marker for viral replication complexes, localized in the extracellular space of infected Nicotiana benthamiana leaves. Previously, we showed that viral RNA is associated with multivesicular bodies (MVBs). Here, using transmission electron microscopy, we observed the proliferation of MVBs during infection and their fusion with the plasma membrane that resulted in the release of their intraluminal vesicles in the extracellular space. Immunogold labeling with a monoclonal antibody that recognizes double-stranded RNA indicated that the released vesicles contained viral RNA. Focused ion beam-extreme high-resolution scanning electron microscopy was used to generate a three-dimensional image that showed extracellular vesicles in the cell wall. The presence of TuMV proteins in the extracellular space was confirmed by proteomic analysis of purified extracellular vesicles from N benthamiana and Arabidopsis (Arabidopsis thaliana). Host proteins involved in biotic defense and in interorganelle vesicular exchange were also detected. The association of extracellular vesicles with viral proteins and RNA emphasizes the implication of the plant extracellular space in viral infection.

Analysis of durum wheat proteome changes under marine and fungal biostimulant treatments using large-scale quantitative proteomics: A useful dataset of durum wheat proteins

Durum wheat requires high nitrogen inputs to obtain the high protein concentration necessary to satisfy pasta and semolina quality criteria. Optimizing plant nitrogen use efficiency is therefore of major importance for wheat grain quality. Here, we studied the impact on grain yield, protein concentration, and for the first time on protein composition of a marine (DPI4913) and a fungal (AF086) biostimulants applied to plant leaves. A large-scale quantitative proteomics analysis of wheat flour samples led to a dataset of 1471 identified proteins. Quantitative analysis of 1391 proteins revealed 26 and 38 proteins with a significantly varying abundance after DPI4913 and AF086 treatment, respectively, with 14 proteins in common. Major effects affected proteins involved in grain technological properties like grain hardness, in storage functions with the gluten protein gamma-gliadin, in regulation processes with transcription regulator proteins, and in stress responses with biotic and abiotic stress defense proteins. The involvement of biostimulants in the abiotic stress response was further suggested by an increase in water-use efficiency for both DPI4913 (15.4%) and AF086 (9.9%) treatments. Overall, our work performed in controlled conditions showed that DPI4913 and AF086 treatments promoted grain yield while maintaining protein concentration, and positively affected protein composition for grain quality.Data are available via ProteomeXchange with identifier PXD012469.

Nectar quality affects ant aggressiveness and biotic defense provided to plants

AbstractAnt–plant mutualisms are useful models for investigating how plant traits mediate interspecific interactions. As plant‐derived resources are essential components of ant diets, plants that offer more nutritious food to ants should be better defended in return, as a result of more aggressive behavior toward natural enemies. We tested this hypothesis in a field experiment by adding artificial nectaries to individuals of the species Vochysia elliptica (Vochysiaceae). Ants were offered one of four liquid foods of different nutritional quality: amino acids, sugar, sugar + amino acids, and water (control). We used live termites (Nasutitermes coxipoensis) as herbivore competitors and observed ant behavior toward them. In 88 hr of observations, we recorded 1,009 interactions with artificial nectaries involving 1,923 individual ants of 26 species. We recorded 381 encounters between ants and termites, of which 38% led to attack. Sixty‐one percent of these attacks led to termite exclusion from the plants. Recruitment and patrolling were highest when ants fed upon nectaries providing sugar + amino acids, the most nutritious food. This increase in recruitment and patrolling led to higher encounter rates between ants and termites, more frequent attacks, and faster and more complete termite removal. Our results are consistent with the hypothesis that plant biotic defense is mediated by resource quality. We highlight the importance of qualitative differences in nectar composition for the outcome of ant–plant interactions.Abstract in Portuguese is available with online material.

Salmonella enterica serovar Typhimurium ATCC 14028S is tolerant to plant defenses triggered by the flagellin receptor FLS2.

ABSTRACTSalmonellosis outbreaks associated with sprouted legumes have been a food safety concern for over two decades. Despite evidence that Salmonella enterica triggers biotic plant defense pathways, it has remained unclear how plant defenses impact Salmonella growth on sprouted legumes. We used Medicago truncatula mutants in which the gene for the flagellin receptor FLS2 was disrupted to demonstrate that plant defenses triggered by FLS2 elicitation do not impact the growth of Salmonella enterica serovar Typhimurium ATCC 14028S. As a control, we tested the growth of Salmonella enterica serovar Typhimurium LT2, which has a defect in rpoS that increases its sensitivity to reactive oxygen species. LT2 displayed enhanced growth on M. truncatula FLS2 mutants in comparison to wild-type M. truncatula. We hypothesize that these growth differences are primarily due to differences in 14028S and LT2 reactive oxygen species sensitivity. Results from this study show that FLS2-mediated plant defenses are ineffective in inhibiting growth of Salmonella entrica 14028S.

Responses of Fuji (Malus domestica) and Shandingzi (Malus baccata) apples to Marssonina coronaria infection revealed by comparative transcriptome analysis

Marssonina leaf blotch of apple caused by Marssonina coronaria is one of the most important diseases of apple (Malus domestica) worldwide. Breeding and the appropriate utilization of disease-resistant varieties is the safest, most effective, most economical and environmentally friendly method for controlling the disease. Previous studies have demonstrated that Shandingzi (Malus baccata) is much more resistant to M. coronaria than Fuji (M. domestica). However, the molecular mechanisms of Fuji and Shandingzi resistance to M. coronaria infection remain unknown. In this study, the responses of Fuji and Shandingzi to M. coronaria infection were studied by comparative transcriptome analysis. As a result, 4898 and 2897 differentially expressed genes (DEGs) in Fuji and Shandingzi, respectively, were identified. Gene Ontology (GO) enrichment analysis of the co-expressed DEGs showed that 25 significant terms were enriched, and the unique DEGs in Shandingzi were mainly associated with the biotic stimulus response (GO: 0009607), defense response (GO: 0006952), stress response (GO: 0006950) and oxidoreductase activity (GO: 0016491). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEGs in both species of Rosaceae were enriched in plant-pathogen interactions, amino acid metabolism and other pathways. In the plant-pathogen interaction pathway, mitogen-activated protein kinase and Ca2+ signaling pathways were induced and enriched in both Shandingzi and Fuji when infected by M. coronaria. Furthermore, the percentages of R genes and disease resistance-related DEGs in Shandingzi were higher than those in Fuji. The qRT-PCR analysis results were consistent with the transcriptome results. The results provide a theoretical basis for breeding disease-resistant varieties.

Methylation-sensitive amplified polymorphism analysis of resistant and susceptible interactions of cucumber with Podosphaera xanthii

Cytosine methylation alterations play key roles in gene regulation in plants interacting with biotrophic pathogens. For resistant and susceptible interactions of cucumber with the powdery mildew pathogen, Podosphaera xanthii, methylation of DNA cytosines were monitored by methylation-sensitive amplified polymorphism (MSAP) analysis from immediately prior to 96 h post inoculation (hpi). In both resistant and susceptible interactions, methylation levels significantly changed over time, but a significant association was observed for decreasing number of loci with demethylated CCGG motif in the susceptible cultivar compared to increasing numbers in the resistant cultivar. Sequencing of six demethylated MSAP fragments from the resistant cultivar and one from susceptible cultivar showed that all contained promoter and cis-acting regulatory elements (CAREs). For the resistant cultivar, three of these were in possible regulatory regions located within or near tRNA coding genes, and those three plus two others contained CAREs associated with response to biotic stress and defense hormones SA, MeJA, ABA and ET. One possible reason for demethylation in the resistant genomic tRNA genes may be that demethylation can de-suppress gene expression resulting in increasing the availability of certain amino acids for defense proteins.

A proteomic approach to seasonal adjustment in the enzyme complement of Korean fir (Abies koreana Wilson) needles

Korean fir is an endemic ornamental tree species facing population decline in Korea. To further understand the acclimatory adjustments it undergoes in response to seasonal extremes, we characterized some of the needle proteins that are upregulated during winter. Two-dimensional gel electrophoresis (2-DE), followed by MALDI TOF/TOF MS/MS and Mascot analyses, was used to visualize changes in protein profiles during acclimation to winter stress. From the 2-DE protein profiles of Korean fir needles, 226 protein spots were detected, many of which accumulated at higher levels during the winter. Among 17 proteins identified, 12 matched proteins associated with photosynthesis and with biotic and abiotic stresses, and eight were significantly upregulated under winter stress. Upregulated proteins included photosynthetic enzymes sedoheptulose-1,7-bisphosphatase and fructose bisphosphate aldolase of the Calvin–Benson cycle, four proteins related to oxidative stress tolerance, two proteins implicated in biotic defense, one heat-shock protein, and five unknown proteins. However, two other oxidative-stress-related proteins were present at high levels throughout the year, and a chitinase and the small subunit of ribulose-1,5-bisphosphate carboxylase showed no seasonal adjustments. Thus, Korean fir needles exhibited winter upregulation of some photosynthetic enzymes, coupled with increased photo protective thermal energy dissipation, and proteins related to abiotic and biotic stress resistance. Winter stress, which can include both low temperature and reduced water availability, in the subalpine region of Mount Halla led to an altered physiological equilibrium with increases in key Calvin–Benson cycle enzymes and increased enzymatic and non-enzymatic protection against oxidative stress.

An Integrative Volatile Terpenoid Profiling and Transcriptomics Analysis for Gene Mining and Functional Characterization of AvBPPS and AvPS Involved in the Monoterpenoid Biosynthesis in Amomum villosum.

Amomum villosum, also known as Fructus Amomi, has been used to treat digestive diseases such as abdominal pain, vomiting, and dysentery. Volatile terpenoids are the active metabolites in the essential oil of Fructus Amomi. Nevertheless, downstream genes responsible for activating metabolites biosynthesis in A. villosum still remain unclear. Here, we report the use of an integrative volatile terpenoid profiling and transcriptomics analysis for mining the corresponding genes involved in volatile terpenoid biosynthesis. Ten terpene synthase (TPS) genes were discovered, and two of them were cloned and functionally characterized. AvTPS1 (AvPS: pinene synthase) catalyzed GPP to form α-pinene and β-pinene; AvTPS3 (AvBPPS: bornyl diphosphate synthase) produced bornyl diphosphate as major product and the other three monoterpenoids as minor products. Metabolite accumulation and gene expression pattern combined with AvPS biochemical characterization suggested that AvPS might play a role in biotic defense. On the other hand, the most active ingredient, bornyl acetate, was highly accumulated in seeds and was consistent with the high expression of AvBPPS, which further indicated that AvBPPS is responsible for the biosynthesis of bornyl acetate, the final metabolite of bornyl diphosphate in A. villosum. This study can be used to improve the quality of A. villosum through metabolic engineering, and for the sustainable production of bornyl acetate in heterologous hosts.

Genome-Wide Identification of MicroRNAs in Response to Cadmium Stress in Oilseed Rape (Brassica napus L.) Using High-Throughput Sequencing.

MicroRNAs (miRNAs) have important roles in regulating stress-response genes in plants. However, identification of miRNAs and the corresponding target genes that are induced in response to cadmium (Cd) stress in Brassica napus remains limited. In the current study, we sequenced three small-RNA libraries from B. napus after 0 days, 1 days, and 3 days of Cd treatment. In total, 44 known miRNAs (belonging to 27 families) and 103 novel miRNAs were identified. A comprehensive analysis of miRNA expression profiles found 39 differentially expressed miRNAs between control and Cd-treated plants; 13 differentially expressed miRNAs were confirmed by qRT-PCR. Characterization of the corresponding target genes indicated functions in processes including transcription factor regulation, biotic stress response, ion homeostasis, and secondary metabolism. Furthermore, we propose a hypothetical model of the Cd-response mechanism in B. napus. Combined with qRT-PCR confirmation, our data suggested that miRNAs were involved in the regulations of TFs, biotic stress defense, ion homeostasis and secondary metabolism synthesis to respond Cd stress in B. napus.

SUMOylome Profiling Reveals a Diverse Array of Nuclear Targets Modified by the SUMO Ligase SIZ1 during Heat Stress.

The posttranslational addition of small ubiquitin-like modifier (SUMO) is an essential protein modification in plants that provides protection against numerous environmental challenges. Ligation is accomplished by a small set of SUMO ligases, with the SAP-MIZ domain-containing SIZ1 and METHYL METHANESULFONATE-SENSITIVE21 (MMS21) ligases having critical roles in stress protection and DNA endoreduplication/repair, respectively. To help identify their corresponding targets in Arabidopsis thaliana, we used siz1 and mms21 mutants for proteomic analyses of SUMOylated proteins enriched via an engineered SUMO1 isoform suitable for mass spectrometric studies. Through multiple data sets from seedlings grown at normal temperatures or exposed to heat stress, we identified over 1000 SUMO targets, most of which are nuclear localized. Whereas no targets could be assigned to MMS21, suggesting that it modifies only a few low abundance proteins, numerous targets could be assigned to SIZ1, including major transcription factors, coactivators/repressors, and chromatin modifiers connected to abiotic and biotic stress defense, some of which associate into multisubunit regulatory complexes. SIZ1 itself is also a target, but studies with mutants protected from SUMOylation failed to uncover a regulatory role. The catalog of SIZ1 substrates indicates that SUMOylation by this ligase provides stress protection by modifying a large array of key nuclear regulators.