The transcription factor TaMYB31 regulates the benzoxazinoid biosynthetic pathway in wheat.

Benzoxazinoids are specialized metabolites that are highly abundant in staple crops, such as maize and wheat. Although their biosynthesis has been studied for several decades, the regulatory mechanisms of the benzoxazinoid pathway remain unknown. Here, we report that the wheat transcription factor MYB31 functions as a regulator of benzoxazinoid biosynthesis genes. A transcriptomic analysis of tetraploid wheat (Triticum turgidum) tissue revealed the up-regulation of two TtMYB31 homoeologous genes upon aphid and caterpillar feeding. TaMYB31 gene silencing in the hexaploid wheat Triticum aestivum significantly reduced benzoxazinoid metabolite levels and led to susceptibility to herbivores. Thus, aphid progeny production, caterpillar body weight gain, and spider mite oviposition significantly increased in TaMYB31-silenced plants. A comprehensive transcriptomic analysis of hexaploid wheat revealed that the TaMYB31 gene is co-expressed with the target benzoxazinoid-encoded Bx genes under several biotic and environmental conditions. Therefore, we analyzed the effect of abiotic stresses on benzoxazinoid levels and discovered a strong accumulation of these compounds in the leaves. The results of a dual fluorescence assay indicated that TaMYB31 binds to the Bx1 and Bx4 gene promoters, thereby activating the transcription of genes involved in the benzoxazinoid pathway. Our finding is the first report of the transcriptional regulation mechanism of the benzoxazinoid pathway in wheat.

The wheat dioxygenase BX6 is involved in the formation of benzoxazinoids in planta and contributes to plant defense against insect herbivores.

Benzoxazinoids are plant specialized metabolites with defense properties, highly abundant in wheat (Triticum), one of the world's most important crops. The goal of our study was to characterize dioxygenase BX6 genes in tetraploid and hexaploid wheat genotypes and to elucidate their effects on defense against herbivores. Phylogenetic analysis revealed four BX6 genes in the hexaploid wheat T. aestivum, but only one ortholog was found in the tetraploid (T. turgidum) wild emmer wheat and the cultivated durum wheat. Transcriptome sequencing of durum wheat plants, damaged by either aphids or caterpillars, revealed that several BX genes, including TtBX6, were upregulated upon caterpillar feeding, relative to the undamaged control plants. A virus-induced gene silencing approach was used to reduce the expression of BX6 in T. aestivum plants, which exhibited both reduced transcript levels and reduced accumulation of different benzoxazinoids. To elucidate the effect of BX6 on plant defense, bioassays with different herbivores feeding on BX6-silenced leaves were conducted. The results showed that plants with silenced BX6 were more susceptible to aphids and the two-spotted spider mite than the control. Overall, our study indicates that wheat BX6 is involved in benzoxazinoid formation in planta and contributes to plant resistance against insect herbivores.

Characterizing serotonin biosynthesis in Setaria viridis leaves and its effect on aphids.

KEY MESSAGE: A combined transcriptomic and metabolic analysis of Setaria viridis leaves responding to aphid infestation was used to identify genes related to serotonin biosynthesis. Setaria viridis (green foxtail), a short life-cycle C4 plant in the Poaceae family, is the wild ancestor of Setaria italica (foxtail millet), a resilient crop that provides good yields in dry and marginal land. Although S. viridis has been studied extensively in the last decade, the molecular mechanisms of insect resistance in this species remain under-investigated. To address this issue, we performed a metabolic analysis of S. viridis and discovered that these plants accumulate the tryptophan-derived compounds tryptamine and serotonin. To elucidate the defensive functions of serotonin, Rhophalosiphum padi (bird cherry-oat aphids) were exposed to this compound, either by exogenous application to the plant medium or with artificial diet bioassays. In both cases, exposure to serotonin increased aphid mortality. To identify genes that are involved in serotonin biosynthesis, we conducted a transcriptome analysis and identified several predicted S. viridis tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H) genes. Two candidate genes were ectopically expressed in Nicotiana tabacum, where SvTDC1 (Sevir.6G066200) had tryptophan decarboxylase activity, and SvT5H1 (Sevir.8G219600) had tryptamine hydroxylase activity. Moreover, the function of the SvTDC1 gene was validated using virus-induced gene silencing in S. italica, which caused a reduction in serotonin levels. This study provides the first evidence of serotonin biosynthesis in Setaria leaves. The biosynthesis of serotonin may play an important role in defense responses and could prove to be useful for developing more pest-tolerant Setaria italica cultivars.

Comparative transcriptomic and metabolic analysis of wild and domesticated wheat genotypes reveals differences in chemical and physical defense responses against aphids.

BACKGROUND: Young wheat plants are continuously exposed to herbivorous insect attack. To reduce insect damage and maintain their growth, plants evolved different defense mechanisms, including the biosynthesis of deterrent compounds named benzoxazinoids, and/or trichome formation that provides physical barriers. It is unclear whether both of these mechanisms are equally critical in providing an efficient defense for wheat seedlings against aphids-an economically costly pest in cereal production. RESULTS: In this study, we compared the transcriptome, metabolome, benzoxazinoids, and trichome density of three selected wheat genotypes, with a focus on differences related to defense mechanisms. We chose diverse wheat genotypes: two tetraploid wheat genotypes, domesticated durum 'Svevo' and wild emmer 'Zavitan,' and one hexaploid bread wheat, 'Chinese Spring.' The full transcriptomic analysis revealed a major difference between the three genotypes, while the clustering of significantly different genes suggested a higher similarity between the two domesticated wheats than between either and the wild wheat. A pathway enrichment analysis indicated that the genes associated with primary metabolism, as well as the pathways associated with defense such as phytohormones and specialized metabolites, were different between the three genotypes. Measurement of benzoxazinoid levels at the three time points (11, 15, and 18 days after germination) revealed high levels in the two domesticated genotypes, while in wild emmer wheat, they were below detection level. In contrast to the benzoxazinoid levels, the trichome density was dramatically higher in the wild emmer than in the domesticated wheat. Lastly, we tested the bird cherry-oat aphid's (Rhopalosiphum padi) performance and found that Chinese Spring is more resistant than the tetraploid genotypes. CONCLUSIONS: Our results show that benzoxazinoids play a more significant defensive role than trichomes. Differences between the abundance of defense mechanisms in the wild and domesticated plants were observed in which wild emmer possesses high physical defenses while the domesticated wheat genotypes have high chemical defenses. These findings provide new insights into the defense adaptations of wheat plants against aphids.

Gut bacteria of weevils developing on plant roots under extreme desert conditions.

BACKGROUND: Many phytophagous insects, whose diet is generally nitrogen-poor, rely on gut bacteria to compensate for nutritional deficits. Accordingly, we hypothesized that insects in desert environments may evolve associations with gut bacteria to adapt to the extremely low nutrient availability. For this, we conducted a systematic survey of bacterial communities in the guts of weevils developing inside mud chambers affixed to plant roots in the Negev Desert of Israel, based on 16S rRNA gene amplicon sequencing. RESULTS: Our analyses revealed that gut bacterial communities in weevil larvae were similar across a wide geographical range, but differed significantly from those of the mud chambers and of the surrounding soils. Nevertheless, a high proportion of bacteria (including all of the core bacteria) found in the weevils were also detected in the mud chambers and soils at low relative abundances. The genus Citrobacter (of the Enterobacteriaceae family) was the predominant group in the guts of all individual weevils. The relative abundance of Citrobacter significantly decreased at the pupal and adult stages, while bacterial diversity increased. A mini literature survey revealed that members of the genus Citrobacter are associated with nitrogen fixation, recycling of uric acid nitrogen, and cellulose degradation in different insects. CONCLUSIONS: The results suggest that although weevils could potentially acquire their gut bacteria from the soil, weevil host internal factors, rather than external environmental factors, were more important in shaping their gut bacterial communities, and suggest a major role for Citrobacter in weevil nutrition in this challenging environment. This study highlights the potential involvement of gut bacteria in the adaptation of insects to nutritional deficiencies under extreme desert conditions.

Cereal aphids differently affect benzoxazinoid levels in durum wheat.

Aphids are major pests in cereal crops that cause direct and indirect damage leading to yield reduction. Despite the fact that wheat provides 20% of the world's caloric and protein diet, its metabolic responses to aphid attack, in general, and specifically its production of benzoxazinoid defense compounds are poorly understood. The objective of this study was to compare the metabolic diversity of durum wheat seedlings (Triticum turgidum ssp. durum) under attack by three different cereal aphids: i) the English grain aphid (Sitobion avenae Fabricius), ii) the bird cherry-oat aphid (Rhopalosiphum padi L.), and iii) the greenbug aphid (Schizaphis graminum Rondani), which are some of the most destructive aphid species to wheat. Insect progeny bioassays and metabolic analyses using chromatography/Q-Exactive/mass spectrometry non-targeted metabolomics and a targeted benzoxazinoid profile were performed on infested leaves. The insect bioassays revealed that the plants were susceptible to S. graminum, resistant to S. avenae, and mildly resistant to R. padi. The metabolic analyses of benzoxazinoids suggested that the predominant metabolites DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin- 3-one) and its glycosylated form DIMBOA-glucoside (Glc) were significantly induced upon both S. avenae, and R. padi aphid feeding. However, the levels of the benzoxazinoid metabolite HDMBOA-Glc (2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside) were enhanced due to the feeding of S. avenae and S. graminum aphids, to which Svevo was the most resistant and the most susceptible, respectively. The results showed a partial correlation between the induction of benzoxazinoids and aphid reproduction. Overall, our observations revealed diverse metabolic responses of wheat seedlings to cereal aphid feeding.

Tripartite symbiosis of plant-weevil-bacteria is a widespread phenomenon in the Negev Desert.

The weevil Conorhynchus palumbus develops in a mud chamber affixed to the roots of the summer annual plant Salsola inermis in the Negev Desert of Israel. The weevil carries nitrogen fixing bacteria, and evidence suggests that plants with weevils utilize the fixed nitrogen. To characterize the distribution, abundance and significance of this unique interaction, we surveyed Salsola plants in 16 sites throughout the Negev Desert. We excavated ~100 plants from each site, recorded the presence of weevils in their roots, and characterized the soil properties in each site. Weevil mud chambers were present in all of the sampled sites and their abundance was positively correlated with soil nitrogen content and with plant size, and negatively correlated with soil grain-size. Intriguingly, we found two additional weevil species-Menecleonus virgatus and Maximus mimosae-residing in mud chambers on Salsola roots, and found one additional Salsola species-S. incanescens-accommodating weevils. Nitrogen fixing bacteria were found in weevil larvae of the two additional species and at multiple sites. Overall, our findings suggest that potentially beneficial associations between weevils and plants may be more common than previously acknowledged, and may play an important role in this desert ecosystem.

Phenylpropanoid Scent Compounds in Petunia x hybrida Are Glycosylated and Accumulate in Vacuoles.

Floral scent has been studied extensively in the model plant Petunia. However, little is known about the intracellular fate of scent compounds. Here, we characterize the glycosylation of phenylpropanoid scent compounds in Petunia x hybrida. This modification reduces scent compounds' volatility, reactivity, and autotoxicity while increasing their water-solubility. Gas chromatography-mass spectrometry (GC-MS) analyses revealed that flowers of petunia cultivars accumulate substantial amounts of glycosylated scent compounds and that their increasing level parallels flower development. In contrast to the pool of accumulated aglycones, which drops considerably at the beginning of the light period, the collective pool of glycosides starts to increase at that time and does not decrease thereafter. The glycoside pool is dynamic and is generated or catabolized during peak scent emission, as inferred from phenylalanine isotope-feeding experiments. Using several approaches, we show that phenylpropanoid scent compounds are stored as glycosides in the vacuoles of petal cells: ectopic expression of Aspergillus niger ß-glucosidase-1 targeted to the vacuole resulted in decreased glycoside accumulation; GC-MS analysis of intact vacuoles isolated from petal protoplasts revealed the presence of glycosylated scent compounds. Accumulation of glycosides in the vacuoles seems to be a common mechanism for phenylpropanoid metabolites.

[ATHLETE'S HEART - THE MORE THE MERRIER?]

INTRODUCTION: Regular moderate exercise training is effective for the prevention and treatment of many common chronic diseases and improves cardiovascular health and life expectancy. However, long-term excessive endurance exercise may induce pathological structural remodeling of the heart. The athlete's heart is characterized by enlargement of cardiac chambers and eccentric hypertrophy with preserved myocardial function as normal physiological adaptations for prolonged and intense endurance physical stress. However, recent studies have demonstrated transient right ventricular dysfunction and elevation of cardiac biomarkers following intense endurance exercise. Repeated bouts of acute stress may cause patchy fibrosis of the right heart and interventricular septum, creating an arrhythmogenic substrate. The following review summarizes the current medical literature on the effects of intense and prolonged endurance exercise on cardiac structure and function and its clinical significance.

The physiologic and histologic properties of the distal internal thoracic artery and its subdivisions.

OBJECTIVE: We compared the flow rates, reactivity, and morphology of the distal internal thoracic artery and its branches, the superior epigastric and musculophrenic arteries, to test their applicability as possible conduits in coronary artery bypass grafting surgeries. METHODS: Skeletonized internal thoracic artery and subdivisions of patients undergoing coronary artery bypass grafting were studied intraoperatively (n = 100) for flow and length measurements and in vitro in organ baths (n = 58) for active response to norepinephrine. Quantitative microscopic analysis of the muscle density and degree of intimal hyperplasia was performed. Results were analyzed according to age, gender, risk factors, and medications. RESULTS: Internal thoracic artery subdivisions contributed an average extra length of 2 cm. Free flow rates were 129 ± 45 mL/min, 114 ± 41 mL/min, and 93 ± 36 mL/min in the internal thoracic artery, superior epigastric artery, and musculophrenic artery, respectively. Sternum and internal thoracic artery length and free flow rates were significantly lower in women. The subdivisions were significantly more reactive to norepinephrine than the distal internal thoracic artery (P ∼ .005), although sensitivity to norepinephrine was similar. Patients treated with beta-blockers had significantly decreased reactivity (P = .009). Microscopic analysis suggests similar muscle content in the internal thoracic artery and subdivisions. Eccentric (28%) and concentric (62%) intimal hyperplasia were observed in 90% of specimens, with no evidence for atherosclerotic plaques. There was no significant difference in the degree of intimal hyperplasia between the distal internal thoracic artery and its subdivisions, and there was no correlation to risk factors. CONCLUSIONS: Our results confirm the previous studies on the higher contractility in internal thoracic artery subdivisions, suggesting caution in the use of the bifurcation for revascularization. However, the extra length, sufficient flow, and favorable histologic properties suggest that the bifurcation may be appropriate for coronary revascularization in selected cases.

The vapB-vapC Operon of Acidovorax citrulli Functions as a Bona-fide Toxin-Antitoxin Module.

Toxin-antitoxin systems are commonly found on plasmids and chromosomes of bacteria and archaea. These systems appear as biscystronic genes encoding a stable toxin and a labile antitoxin, which protects the cells from the toxin's activity. Under specific, mostly stressful conditions, the unstable antitoxin is degraded, the toxin becomes active and growth is arrested. Using genome analysis we identified a putative toxin-antitoxin encoding system in the genome of the plant pathogen Acidovorax citrulli. The system is homologous to vapB-vapC systems from other bacterial species. PCR and phylogenetic analyses suggested that this locus is unique to group II strains of A. citrulli. Using biochemical and molecular analyses we show that A. citrulli VapBC module is a bona-fide toxin-antitoxin module in which VapC is a toxin with ribonuclease activity that can be counteracted by its cognate VapB antitoxin. We further show that transcription of the A. citrulli vapBC locus is induced by amino acid starvation, chloramphenicol and during plant infection. Due to the possible role of TA systems in both virulence and dormancy of human pathogenic bacteria, studies of these systems are gaining a lot of attention. Conversely, studies characterizing toxin-antitoxin systems in plant pathogenic bacteria are lacking. The study presented here validates the activity of VapB and VapC proteins in A. citrulli and suggests their involvement in stress response and host-pathogen interactions.