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.     

Soybean Resistance to Phakopsora pachyrhizi as Affected by Acibenzolar‐S‐Methyl,Jasmonic Acid and Silicon

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is one of the most important diseases on soybean. At the moment, ASR is managed mainly with fungicides due to the absence of commercial cultivars with resistance to this disease. This study evaluated the effects of acibenzolar‐Smethyl (ASM), jasmonic acid (JA), potassium silicate (PS) and calcium silicate (CS) on soybean resistance to ASR. The ASM, JA and PS were sprayed to leaves 24 h prior to inoculation with P. pachyrhizi. The CS was amended to the soil. The incubation period (time from the inoculation until symptoms development) was longer for plants growing in soil amended with CS or sprayed with ASM in comparison with plants sprayed with water (control). Plants sprayed with ASM had longer latent period (time from the inoculation until signs appearance) in comparison with the control plants. Plants sprayed with PS showed fewer uredia per cm² of leaf in relation to the control plants. The ASM and PS were the most effective treatments in reducing the ASR symptoms in contrast to the JA and CS treatments. The JA served as an inducer of susceptibility to ASR.     

Defence‐Related Enzymes in Soybean Resistance to Target Spot

Target spot, caused by the fungus Corynespora cassiicola, has become a serious foliar disease in soybean production in the Brazilian Cerrado. Information in the literature regarding the biochemical defence responses of soybean to C. cassiicola infection is rare. Therefore, the objective of this study was to determine the biochemical features associated with soybean resistance to target spot. The activities of chitinases (CHI), β‐1‐3‐glucanases (GLU), phenylalanine ammonia‐lyases (PAL), peroxidases (POX), polyphenol oxidases (PPO) and lipoxygenases (LOX), as well as the concentrations of total soluble phenolics (TSP) and lignin‐thioglycolic acid (LTGA) derivatives, were determined in soybean leaves from both a resistant (FUNDACEP 59) and a susceptible (TMG 132) cultivar. The target spot severity, number of lesions per cm² of leaflet and area under the disease progress curve were significantly lower for plants from cv. FUNDACEP 59 compared to plants from cv. TMG 132. The GLU, CHI, PAL, POX and PPO activities and the concentration of LTGA derivatives increased significantly, whereas LOX activity decreased significantly on the leaves infected by C. cassiicola. Inoculated plants from cv. FUNDACEP 59 showed a higher PPO activity and concentrations of TSP and LTGA derivatives at 4 and 6 days after inoculation compared to plants from cv. TMG 132. In conclusion, the results of this study demonstrated that the defence‐related enzyme activities increased upon C. cassiicola infection, regardless of the basal level of resistance of the cultivar studied. The increases in PPO activity and concentrations of TSP and LTGA derivatives, but lower LOX activity, at early stages of C. cassiicola infection were highly associated with soybean resistance to target spot.     

Construction and comparison of three reference‐quality genome assemblies for soybean

We report reference‐quality genome assemblies and annotations for two accessions of soybean (Glycine max) and for one accession of Glycine soja, the closest wild relative of G. max. The G. max assemblies provided are for widely used US cultivars: the northern line Williams 82 (Wm82) and the southern line Lee. The Wm82 assembly improves the prior published assembly, and the Lee and G. soja assemblies are new for these accessions. Comparisons among the three accessions show generally high structural conservation, but nucleotide difference of 1.7 single‐nucleotide polymorphisms (snps) per kb between Wm82 and Lee, and 4.7 snps per kb between these lines and G. soja. snp distributions and comparisons with genotypes of the Lee and Wm82 parents highlight patterns of introgression and haplotype structure. Comparisons against the US germplasm collection show placement of the sequenced accessions relative to global soybean diversity. Analysis of a pan‐gene collection shows generally high conservation, with variation occurring primarily in genomically clustered gene families. We found approximately 40–42 inversions per chromosome between either Lee or Wm82v4 and G. soja, and approximately 32 inversions per chromosome between Wm82 and Lee. We also investigated five domestication loci. For each locus, we found two different alleles with functional differences between G. soja and the two domesticated accessions. The genome assemblies for multiple cultivated accessions and for the closest wild ancestor of soybean provides a valuable set of resources for identifying causal variants that underlie traits for the domestication and improvement of soybean, serving as a basis for future research and crop improvement efforts for this important crop species.     

Silicon‐Induced Changes in the Antioxidant System Reduce Soybean Resistance to Frogeye Leaf Spot

Frogeye leaf spot (FLS), caused by the fungus Cercospora sojina, is one of the most important soybean diseases and can cause great yield losses. Several studies have demonstrated that silicon (Si) enhances the plant antioxidant system, especially when they are subjected to stresses. Thus, this study was designed to evaluate the effect of Si on soybean resistance to FLS, on the antioxidant system, on the concentration of reactive oxygen species and on cellular damage during the infection process of C. sojina. Plants from cultivars Bossier and Conquista, susceptible and resistant to FLS, respectively, were supplied with either 0 (?Si) or 2 mm (+Si) and non‐inoculated or inoculated with C. sojina. FLS severity was greater for Bossier than for Conquista, regardless of the Si supply, and it was increased by Si for both cultivars. The activities of the most antioxidant enzymes were lower in the +Si plants than in the ?Si plants when they were not inoculated. Inoculated plants usually showed an increased enzyme activities and higher concentrations of ascorbate and reduced glutathione than did the non‐inoculated plants, regardless of Si supply. At advanced stages of fungal infection, the +Si‐inoculated plants from Bossier had higher activity of most antioxidant enzymes and higher concentrations of superoxide and malondialdehyde compared to the non‐inoculated plants as a result of an increased oxidative stress. The results from this study provide the first evidence that Si reduces the basal activity of antioxidant enzymes in soybean leaves leading to an increase in host susceptibility to FLS.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

ICAM‐1 and MKL‐1 polymorphisms impose considerable impacts on coronary heart disease occurrence

This study was aimed to explore the correlation of intercellular adhesion molecule‐1 (ICAM‐1) K469E and megakaryoblastic leukaemia factor‐1 (MKL‐1) ?184C/T polymorphisms with the susceptibility to coronary heart disease (CHD) in the Chinese Han population. 100 CHD patients and 91 healthy people that had no blood connection with each other were enrolled in this case‐control study. ICAM‐1 and MKL‐1 polymorphisms were genotyped by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) approach. Multiple logistic regression was used to analyse the correlation between polymorphisms of ICAM‐1 and MKL‐1 and CHD susceptibility. Differences of genotype and allele frequencies of the two SNPs between case and control groups were analysed by chi‐square test. Odds ratios (ORs) and 95% confidence intervals (CIs) were indicated relative susceptibility of CHD. The distributions of ICAM‐1 and MKL‐1 polymorphisms in each group conformed to Hardy‐Weinberg equilibrium (HWE). After adjusting for traditional risk factors, the TT genotype frequency of MKL‐1 ?184C/T polymorphism was found significantly higher in case group than in control group (P < .05). Meanwhile, T allele frequency increased in case group compared with control group, and the differences had statistical significance (P = .04, OR = 2.34, 95% CI = 1.34‐5.26). Logistic regression analysis in this study proved that smoking, hypertension, diabetes and triglyceride (TG) were all risk factors for CHD ICAM‐1 K469E polymorphism has no association with the onset of CHD. But MKL‐1 ?184C/T polymorphism is associated with the risk of CHD and T allele might be a susceptibility factor for CHD.     

Global investigation of the co‐evolution of MIRNA genes and microRNA targets during soybean domestication

Although the selection of coding genes during plant domestication has been well studied, the evolution of MIRNA genes (MIRs) and the interaction between microRNAs (miRNAs) and their targets in this process are poorly understood. Here, we present a genome‐wide survey of the selection of MIRs and miRNA targets during soybean domestication and improvement. Our results suggest that, overall, MIRs have higher evolutionary rates than miRNA targets. Nonetheless, they do demonstrate certain similar evolutionary patterns during soybean domestication: MIRs and miRNA targets with high expression and duplication status, and with greater numbers of partners, exhibit lower nucleotide divergence than their counterparts without these characteristics, suggesting that expression level, duplication status, and miRNA–target interaction are essential for evolution of MIRs and miRNA targets. Further investigation revealed that miRNA–target pairs that are subjected to strong purifying selection have greater similarities than those that exhibited genetic diversity. Moreover, mediated by domestication and improvement, the similarities of a large number of miRNA–target pairs in cultivated soybean populations were increased compared to those in wild soybeans, whereas a small number of miRNA–target pairs exhibited decreased similarity, which may be associated with the adoption of particular domestication traits. Taken together, our results shed light on the co‐evolution of MIRs and miRNA targets during soybean domestication.     

Spaceflight‐induced enhancement of 2‐keto‐L‐gulonic acid production by a mixed culture of Ketogulonigenium vulgare and Bacillus thuringiensis

Two bacterial strains used for industrial production of 2‐keto‐L‐gulonic acid (2‐KLG), Ketogulonigenium vulgare 2 and Bacillus thuringiensis 1514, were loaded onto the spacecraft Shenzhou VII and exposed to space conditions for 68 h in an attempt to increase their fermentation productivities of 2‐KLG. An optimal combination of mutants B. thuringiensis 320 and K. vulgare 2194 (KB2194‐320) was identified by systematically screening the pH and 2‐KLG production of 16 000 colonies. Compared with the coculture of parent strains, the conversion rate of L‐sorbose to 2‐KLG by KB2194‐320 in shake flask fermentation was increased significantly from 82·7% to 95·0%. Furthermore, a conversion rate of 94·5% and 2‐KLG productivity of 1·88 g l^?1 h^?1 were achieved with KB2194‐320 in industrial‐scale fermentation (260 m³ fermentor). An observed increase in cell number of K2194 (increased by 47·8%) during the exponential phase and decrease in 2‐KLG reductase activity (decreased by 46·0%) were assumed to explain the enhanced 2‐KLG production. The results suggested that the mutants KB2194‐320 could be ideal substitutes for the currently employed strains in the 2‐KLG fermentation process and demonstrated the feasibility of using spaceflight to breed high‐yielding 2‐KLG‐producing strains for vitamin C production.

Significance and Impact of the Study

KB2194‐320, a combination of two bacterial strains bred by spaceflight mutation, exhibited significantly improved 2‐KLG productivity and hence could potentially increase the efficiency and reduce the cost of vitamin C production by the two‐step fermentation process. In addition, a new pH indicator method was applied for rational screening of K2, which dramatically improved the efficiency of screening.     

Sub‐lethal UV‐C radiation induces callose,hydrogen peroxide and defence‐related gene expression in Arabidopsis thaliana

Exposure of plants to UV‐C irradiation induces gene expression and cellular responses that are commonly associated with wounding and pathogen defence, and in some cases can lead to increased resistance against pathogen infection. We examined, at a physiological, molecular and biochemical level, the effects of and responses to, sub‐lethal UV‐C exposure on Arabidopsis plants when irradiated with increasing dosages of UV‐C radiation. Following UV‐C exposure plants had reduced leaf areas over time, with the severity of reduction increasing with dosage. Severe morphological changes that included leaf glazing, bronzing and curling were found to occur in plants treated with the 1000 J·m^?2 dosage. Extensive damage to the mesophyll was observed, and cell death occurred in both a dosage‐ and time‐dependent manner. Analysis of H₂O₂ activity and the pathogen defence marker genes PR1 and PDF1.2 demonstrated induction of these defence‐related responses at each UV‐C dosage tested. Interestingly, in response to UV‐C irradiation the production of callose (β‐1,3‐glucan) was identified at all dosages examined. Together, these results show plant responses to UV‐C irradiation at much lower doses than have previously been reported, and that there is potential for the use of UV‐C as an inducer of plant defence.     

Utilization of UPLC/Q‐TOF‐MS‐Based Metabolomics and AFLP‐Based Marker‐Assisted Selection to Facilitate/Assist Conventional Breeding of Polygala tenuifolia

As one of the most important traditional Chinese medicine, the quality of Polygala tenuifolia is difficult to control and a new method must be established to facilitate/assist the breeding of P. tenuifolia. In this study, UPLC/Q‐TOF‐MS‐based metabolomics analysis was performed to determine the chemical composition and screen metabolite biomarkers according to agronomic traits. A total of 29 compounds and 18 metabolite biomarkers were found. AFLP‐based marker‐assisted selection (MAS) was used to identify molecular marker bands and screen characteristic bands associated with specific agronomic traits. 184 bands and 76 characteristic AFLP bands were found. The correlation network between compounds and characteristic AFLP bands was built, so we may directly breed certain P. tenuifolia herbs with special agronomic traits (or characteristic AFLP bands), which exhibit specific pharmacological functions depending on the content of the active compounds. The proposed method of metabolomics coupled with MAS could facilitate/assist the breeding of P. tenuifolia.     

lac‐1 and lag‐1 with ras‐1 affect aging and the biological clock in Neurospora crassa

Using an automated cell counting technique developed previously (Case et al., Ecology and Evolution 2014; 4: 3494), we explore the lifespan effects of lac‐1, a ceramide synthase gene paralogous to lag‐1 in Neurospora crassa in conjunction with the band bd (ras‐1) gene. We find that the replicative lifespan of a lac‐1^KO bd double mutants is short, about one race tube cycle, and this double mutant lacks a strong ~21‐hr clock cycle as shown by race tube and fluorometer analysis of fluorescent strains including lac‐1^KO. This short replicative lifespan phenotype is contrasted with a very long estimated chronological lifespan for lac‐1^KO bd double mutants from 247 to 462 days based on our regression analyses on log viability, and for the single mutant lac‐1^KO, 161 days. Both of these estimated lifespans are much higher than that of previously studied WT and bd single mutant strains. In a lac‐1 rescue and induction experiment, the expression of lac‐1⁺ as driven by a quinic acid‐dependent promoter actually decreases the median chronological lifespan of cells down to only 7 days, much lower than the 34‐day median lifespan found in control bd conidia also grown on quinic acid media, which we interpret as an effect of balancing selection acting on ceramide levels based on previous findings from the literature. Prior work has shown phytoceramides can act as a signal for apoptosis in stressed N. crassa cells. To test this hypothesis of balancing selection on phytoceramide levels, we examine the viability of WT, lag‐1^KO bd, and lac‐1^KO bd strains following the dual stresses of heat and glycolysis inhibition, along with phytoceramide treatments of different dosages. We find that the phytoceramide dosage–response curve is altered in the lag‐1^KO bd mutant, but not in the lac‐1^KO bd mutant. We conclude that phytoceramide production is responsible for the previously reported longevity effects in the lag‐1^KO bd mutant, but a different ceramide may be responsible for the longevity effect observed in the lac‐1^KO bd mutant.