In vivo detection of protein cysteine sulfenylation in plastids

Protein cysteine thiols are post‐translationally modified under oxidative stress conditions. Illuminated chloroplasts are one of the important sources of hydrogen peroxide (H₂O₂) and are highly sensitive to environmental stimuli, yet a comprehensive view of the oxidation‐sensitive chloroplast proteome is still missing. By targeting the sulfenic acid YAP1C‐trapping technology to the plastids of light‐grown Arabidopsis cells, we identified 132 putatively sulfenylated plastid proteins upon H₂O₂ pulse treatment. Almost half of the sulfenylated proteins are enzymes of the amino acid metabolism. Using metabolomics, we observed a reversible decrease in the levels of the amino acids Ala, Asn, Cys, Gln, Glu, His, Ile, Leu, Lys, Phe, Ser, Thr and Val after H₂O₂ treatment, which is in line with an anticipated decrease in the levels of the glycolysis and tricarboxylic acid metabolites. Through the identification of an organelle‐tailored proteome, we demonstrated that the subcellular targeting of the YAP1C probe enables us to study in vivo cysteine sulfenylation at the organellar level. All in all, the identification of these oxidation events in plastids revealed that several enzymes of the amino acid metabolism rapidly undergo cysteine oxidation upon oxidative stress.     

A genomic investigation of ecological differentiation between free‐living and Drosophila‐associated bacteria

Various bacterial taxa have been identified both in association with animals and in the external environment, but the extent to which related bacteria from the two habitat types are ecologically and evolutionarily distinct is largely unknown. This study investigated the scale and pattern of genetic differentiation between bacteria of the family Acetobacteraceae isolated from the guts of Drosophila fruit flies, plant material and industrial fermentations. Genome‐scale analysis of the phylogenetic relationships and predicted functions was conducted on 44 Acetobacteraceae isolates, including newly sequenced genomes from 18 isolates from wild and laboratory Drosophila. Isolates from the external environment and Drosophila could not be assigned to distinct phylogenetic groups, nor are their genomes enriched for any different sets of genes or category of predicted gene functions. In contrast, analysis of bacteria from laboratory Drosophila showed they were genetically distinct in their universal capacity to degrade uric acid (a major nitrogenous waste product of Drosophila) and absence of flagellar motility, while these traits vary among wild Drosophila isolates. Analysis of the competitive fitness of Acetobacter discordant for these traits revealed a significant fitness deficit for bacteria that cannot degrade uric acid in culture with Drosophila. We propose that, for wild populations, frequent cycling of Acetobacter between Drosophila and the external environment prevents genetic differentiation by maintaining selection for traits adaptive in both the gut and external habitats. However, laboratory isolates bear the signs of adaptation to persistent association with the Drosophila host under tightly defined environmental conditions.     

Stacking of a stearoyl‐ACP thioesterase with a dual‐silenced palmitoyl‐ACP thioesterase and ∆12 fatty acid desaturase in transgenic soybean

Soybean (Glycine max (L.) Merr) is valued for both its protein and oil, whose seed is composed of 40% and 20% of each component, respectively. Given its high percentage of polyunsaturated fatty acids, linoleic acid and linolenic acid, soybean oil oxidative stability is relatively poor. Historically food processors have employed a partial hydrogenation process to soybean oil as a means to improve both the oxidative stability and functionality in end‐use applications. However, the hydrogenation process leads to the formation of trans‐fats, which are associated with negative cardiovascular health. As a means to circumvent the need for the hydrogenation process, genetic approaches are being pursued to improve oil quality in oilseeds. In this regard, we report here on the introduction of the mangosteen (Garcinia mangostana) stearoyl‐ACP thioesterase into soybean and the subsequent stacking with an event that is dual‐silenced in palmitoyl‐ACP thioesterase and ?12 fatty acid desaturase expression in a seed‐specific fashion. Phenotypic analyses on transgenic soybean expressing the mangosteen stearoyl‐ACP thioesterase revealed increases in seed stearic acid levels up to 17%. The subsequent stacked with a soybean event silenced in both palmitoyl‐ACP thioesterase and ?12 fatty acid desaturase activity, resulted in a seed lipid phenotype of approximately 11%–19% stearate and approximately 70% oleate. The oil profile created by the stack was maintained for four generations under greenhouse conditions and a fifth generation under a field environment. However, in generation six and seven under field conditions, the oleate levels decreased to 30%–40%, while the stearic level remained elevated.     

Isomerization of octadecapentaenoic acid (18:5n‐3) in algal lipid samples under derivatization for GC and GC‐MS analysis

During gas chromatography (GC) analysis of fatty acid (FA) composition of the dinoflagellate Gymnodinium kowalevskii, we found unex‐pectedly low and irreproducible content of all‐cis‐3,6,9,12,15‐octadecapentaenoic acid (18:5n‐3), which is an important chemotaxonomic marker of several classes of microalgae. We compared chromatographic behavior of 18:5n‐3 methyl ester and other GC derivatives obtained using different conventional methods of derivatization. The use of methods based on saponification or base‐catalyzed transesterification resulted in a mixture of double‐bond positional isomers of 18:5. On a SUPELCOWAX 10 column, the equivalent chain length (ECL) value for authentic 18:5n‐3 methyl ester was 20.22, whereas the main component after base‐catalyzed methylation had ECL 20.88. Attempts to prepare N‐acyl pyrrolidides or 4,4‐dimethyloxazoline (DMOX) derivatives of 18:5n‐3 also gave inadequate results. These derivatives also showed a main peak corresponding to isomerized 18:5. Mass spectra for both DMOX and pyrrolidide derivatives of this compound showed the base peak at m/z 139, probably corresponding to 2,6,9,12,15‐18:5 acid. Of all methods tested for methylation, only derivatization with 5% HCl or 1% sulphuric acid in methanol gave satisfactory results. Therefore, GC or GC‐mass spectrometry analyses of algal lipids containing 18:5n‐3 may be inaccurate when base‐catalyzed methods of FA derivatization are applied. The best and simplest way to avoid incorrect GC results is to use standard acid‐catalyzed methylation.     

Salivary proteins of spider mites suppress defenses in Nicotiana benthamiana and promote mite reproduction

Spider mites (Tetranychidae sp.) are widely occurring arthropod pests on cultivated plants. Feeding by the two‐spotted spider mite T. urticae, a generalist herbivore, induces a defense response in plants that mainly depends on the phytohormones jasmonic acid and salicylic acid (SA). On tomato (Solanum lycopersicum), however, certain genotypes of T. urticae and the specialist species T. evansi were found to suppress these defenses. This phenomenon occurs downstream of phytohormone accumulation via an unknown mechanism. We investigated if spider mites possess effector‐like proteins in their saliva that can account for this defense suppression. First we performed an in silico prediction of the T. urticae and the T. evansi secretomes, and subsequently generated a short list of candidate effectors based on additional selection criteria such as life stage‐specific expression and salivary gland expression via whole mount in situ hybridization. We picked the top five most promising protein families and then expressed representatives in Nicotiana benthamiana using Agrobacterium tumefaciens transient expression assays to assess their effect on plant defenses. Four proteins from two families suppressed defenses downstream of the phytohormone SA. Furthermore, T. urticae performance on N. benthamiana improved in response to transient expression of three of these proteins and this improvement was similar to that of mites feeding on the tomato SA accumulation mutant nahG. Our results suggest that both generalist and specialist plant‐eating mite species are sensitive to SA defenses but secrete proteins via their saliva to reduce the negative effects of these defenses.     

Overexpression of AtPAD4 in transgenic Brachypodium distachyon enhances resistance to Puccinia brachypodii

• Brachypodium distachyon (L.) has recently emerged as a model for temperate grasses for investigating the molecular basis of plant–pathogen interactions. Phytoalexin deficient 4 (PAD4) plays a regulatory role in mediating expression of genes involved in plant defence.
• In this research, we generated transgenic B. distachyon plants constitutively overexpressing AtPAD4. Two transgenic B. distachyon lines were verified using PCR and GUS phenotype.
• Constitutive expression of AtPAD4 in B. distachyon enhanced resistance to Puccinia brachypodii. P. brachypodii generated less urediniospores on transgenic than on wild‐type plants. AtPAD4 overexpression enhanced salicylic acid (SA) levels in B. distachyon‐infected tissues. qRT‐PCR showed that expression of pathogenesis‐related 1 (PR1) and other defence‐related genes were up‐regulated in transformed B. distachyon following infection with P. brachypodii.
• Our results indicate that AtPAD4 overexpression in B. distachyon plants led to SA accumulation and induced PR gene expression that reduced the rate of colonisation by P. brachypodii.

     

The oxalyl‐CoA synthetase‐regulated oxalate and its distinct effects on resistance to bacterial blight and aluminium toxicity in rice

• Oxalic acid is widely distributed in biological systems and known to play functional roles in plants. The gene AAE3 was recently identified to encode an oxalyl‐CoA synthetase (OCS) in Arabidopsis that catalyses the conversion of oxalate and CoA into oxalyl‐CoA. It will be particularly important to characterise the homologous gene in rice since rice is not only a monocotyledonous model plant, but also a staple food crop.
• Various enzymatic and biological methods have been used to characterise the homologous gene.
• We first defined that AAE3 in the rice genome (OsAAE3) also encodes an OCS enzyme. Its K_m for oxalate is 1.73 ± 0.12 mm , and V_m is 6824.9 ± 410.29 U·min^?1·mg protein^?1. Chemical modification and site‐directed mutagenesis analyses identified thiols as the active site residues for rice OCS catalysis, suggesting that the enzyme might be regulated by redox state. Subcellular localisation assay showed that the enzyme is located in the cytosol and predominantly distributed in leaf epidermal cells. As expected, oxalate levels increased when OCS was suppressed in RNAi transgenic plants. More interestingly, OCS‐suppressed plants were more susceptible to bacterial blight but more resistant to Al toxicity.
• The results demonstrate that the OsAAE3‐encoded protein also acts as an OCS in rice, and may play different roles in coping with stresses. These molecular, enzymatic and functional data provide first‐hand information to further clarify the function and mechanism of OCS in rice plants.

     

Control of resin production in Araucaria angustifolia,an ancient South American conifer

Araucaria angustifolia is an ancient slow‐growing conifer that characterises parts of the Southern Atlantic Forest biome, currently listed as a critically endangered species. The species also produces bark resin, although the factors controlling its resinosis are largely unknown. To better understand this defence‐related process, we examined the resin exudation response of A. angustifolia upon treatment with well‐known chemical stimulators used in fast‐growing conifers producing both bark and wood resin, such as Pinus elliottii. The initial hypothesis was that A. angustifolia would display significant differences in the regulation of resinosis. The effect of Ethrel^® (ET – ethylene precursor), salicylic acid (SA), jasmonic acid (JA), sulphuric acid (SuA) and sodium nitroprusside (SNP – nitric oxide donor) on resin yield and composition in young plants of A. angustifolia was examined. In at least one of the concentrations tested, and frequently in more than one, an aqueous glycerol solution applied on fresh wound sites of the stem with one or more of the adjuvants examined promoted an increase in resin yield, as well as monoterpene concentration (α‐pinene, β‐pinene, camphene and limonene). Higher yields and longer exudation periods were observed with JA and ET, another feature shared with Pinus resinosis. The results suggest that resinosis control is similar in Araucaria and Pinus. In addition, A. angustifolia resin may be a relevant source of valuable terpene chemicals, whose production may be increased by using stimulating pastes containing the identified adjuvants.