Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence.

Small RNAs (sRNAs) are essential for normal plant development and range in size classes of 21-24 nucleotides. The 22nt small interfering RNAs (siRNAs) and miRNAs are processed by Dicer-like 2 (DCL2) and DCL1 respectively and can initiate secondary siRNA production from the target transcript. 22nt siRNAs are under-represented due to competition between DCL2 and DCL4, while only a small number of 22nt miRNAs exist. Here we produce abundant 22nt siRNAs and other siRNA size classes using long hairpin RNA (hpRNA) transgenes. By introducing asymmetric bulges into the antisense strand of hpRNA, we shifted the dominant siRNA size class from 21nt of the traditional hpRNA to 22, 23 and 24nt of the asymmetric hpRNAs. The asymmetric hpRNAs effectively silenced a ß-glucuronidase (GUS) reporter transgene and the endogenous ethylene insensitive-2 (EIN2) and chalcone synthase (CHS) genes. Furthermore, plants containing the asymmetric hpRNA transgenes showed increased amounts of 21nt siRNAs downstream of the hpRNA target site compared to plants with the traditional hpRNA transgenes. This indicates that these asymmetric hpRNAs are more effective at inducing secondary siRNA production to amplify silencing signals. The 22nt asymmetric hpRNA constructs enhanced virus resistance in plants compared to the traditional hpRNA constructs.

G-U base-paired hpRNA confers potent inhibition of small RNA function in plants.

MicroRNA (miRNA) target mimicry technologies, utilizing naturally occurring miRNA decoy molecules, represent a potent tool for analyzing miRNA function. In this study, we present a highly efficient small RNA (sRNA) target mimicry design based on G-U base-paired hairpin RNA (hpG:U), which allows for the simultaneous targeting of multiple sRNAs. The hpG:U constructs consistently generate high amounts of intact, polyadenylated stem-loop (SL) RNA outside the nuclei, in contrast to traditional hairpin RNA designs with canonical base pairing (hpWT), which were predominantly processed resulting in a loop. By incorporating a 460-bp G-U base-paired double-stranded stem and a 312-576 nt loop carrying multiple miRNA target mimicry sites (GUMIC), the hpG:U construct displayed effective repression of three Arabidopsis miRNAs, namely miR165/166, miR157, and miR160, both individually and in combination. Additionally, a GUMIC construct targeting a prominent cluster of siRNAs derived from cucumber mosaic virus (CMV) Y-satellite RNA (Y-Sat) effectively inhibited Y-Sat siRNA-directed silencing of the chlorophyll biosynthetic gene CHLI, thereby reducing the yellowing symptoms in infected Nicotiana plants. Therefore, the G-U base-paired hpRNA, characterized by differential processing compared to traditional hpRNA, acts as an efficient decoy for both miRNAs and siRNAs. This technology holds great potential for sRNA functional analysis and the management of sRNA-mediated diseases.

Umbilical artery cord blood glucose predicted hypoglycemia in gestational diabetes mellitus and other at-risk newborns.

BACKGROUND: To explore the value of umbilical artery cord blood glucose (UACBG) in predicting hypoglycemia in gestational diabetes mellitus (GDM) and other at-risk newborns, and to provide a cut-off UACBG value for predicting hypoglycemia occurrence. METHODS: In this prospective study, we enrolled at-risk infants delivered vaginally, including neonates born to mothers with GDM, premature, macrosomic, and low birth weight. We separated the infants into GDM group and other at-risk group. All subjects underwent UACBG measurement during delivery. Neonatal peripheral blood glucose measurement was performed at 0.5 and 2 h after birth. The predictive performance of UACBG for neonatal hypoglycemia was assessed using receiver operating characteristic curve (ROC), area under curve (AUC), sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV). RESULTS: 916 newborns were included, with 538 in GDM group and 378 in other at-risk group. 85 neonates were diagnosed hypoglycemia within 2 h after birth, including 36 belonging to GDM group and 49 to other at-risk group. For hypoglycemia prediction within 2 h, the best cut-off of UACBG was 4.150 mmol/L, yielding an AUC of 0.688 (95% CI 0.625-0.751) and a NPV of 0.933. In detail, the AUC was 0.680 in GDM group (95% CI 0.589-0.771), with the optimal cut-off of 4.150 mmol/L and a NPV of 0.950. In other at-risk group, the AUC was 0.678(95% CI 0.586-0.771), the best threshold was 3.950 mmol/L and the NPV was 0.908. No significant differences were observed between GDM group and other at-risk group in AUC at 0.5 h, 2 h and within 2 h. CONCLUSIONS: UACBG has a high NPV for predicting neonatal hypoglycemia within 2 h after birth. It was implied that individuals with cord blood glucose levels above the threshold were at lower risk for hypoglycemia. UACBG monitoring provides evidence for subsequent classified management of hypoglycemia.

Spectrum Sensing Method Based on Residual Dense Network and Attention.

To address the problems of gradient vanishing and limited feature extraction capability of traditional CNN spectrum sensing methods in deep network structures and to effectively avoid network degradation issues under deep network structures, this paper proposes a collaborative spectrum sensing method based on Residual Dense Network and attention mechanisms. This method involves stacking and normalizing the time-domain information of the signal, constructing a two-dimensional matrix, and mapping it to a grayscale image. The grayscale images are divided into training and testing sets, and the training set is used to train the neural network to extract deep features. Finally, the test set is fed into the well-trained neural network for spectrum sensing. Experimental results show that, under low signal-to-noise ratios, the proposed method demonstrates superior spectral sensing performance compared to traditional collaborative spectrum sensing methods.

Can lymphovascular invasion be predicted by contrast-enhanced CT imaging features in patients with esophageal squamous cell carcinoma? A preliminary retrospective study.

BACKGROUND: To investigate the value of contrast-enhanced CT (CECT)-derived imaging features in predicting lymphovascular invasion (LVI) status in esophageal squamous cell carcinoma (ESCC) patients. METHODS: One hundred and ninety-seven patients with postoperative pathologically confirmed esophageal squamous cell carcinoma treated in our hospital between January 2017 and January 2019 were enrolled in our study, including fifty-nine patients with LVI and one hundred and thirty-eight patients without LVI. The CECT-derived imaging features of all patients were analyzed. The CECT-derived imaging features were divided into quantitative features and qualitative features. The quantitative features consisted of the CT attenuation value of the tumor (CTVTumor), the CT attenuation value of the normal esophageal wall (CTVNormal), the CT attenuation value ratio of the tumor-to-normal esophageal wall (TNR), the CT attenuation value difference between the tumor and normal esophageal wall (ΔTN), the maximum thickness of the tumor measured by CECT (Thickness), the maximum length of the tumor measured by CECT (Length), and the gross tumor volume measured by CECT (GTV). The qualitative features consisted of an enhancement pattern, tumor margin, enlarged blood supply or drainage vessels to the tumor (EVFDT), and tumor necrosis. For the clinicopathological characteristics and CECT-derived imaging feature analysis, the chi-squared test was used for categorical variables, the Mann-Whitney U test was used for continuous variables with a nonnormal distribution, and the independent sample t-test was used for the continuous variables with a normal distribution. The trend test was used for ordinal variables. The association between LVI status and CECT-derived imaging features was analyzed by univariable logistic analysis, followed by multivariable logistic regression and receiver operating characteristic (ROC) curve analysis. RESULTS: The CTVTumor, TNR, ΔTN, Thickness, Length, and GTV in the group with LVI were higher than those in the group without LVI (P < 0.05). A higher proportion of patients with heterogeneous enhancement pattern, irregular tumor margin, EVFDT, and tumor necrosis were present in the group with LVI (P < 0.05). As revealed by the univariable logistic analysis, the CECT-derived imaging features, including CTVTumor, TNR, ΔTN and enhancement pattern, Thickness, Length, GTV, tumor margin, EVFDT, and tumor necrosis were associated with LVI status (P < 0.05). Only the TNR (OR 8.655; 95% CI 2.125-37.776), Thickness (OR 6.531; 95% CI 2.410-20.608), and tumor margin (OR 4.384; 95% CI 2.004-9.717) were independent risk factors for LVI in the multivariable logistic regression analysis. The ROC curve analysis incorporating the above three CECT-derived imaging features showed that the area under the curve obtained by the multivariable logistic regression model was 0.820 (95% CI 0.754-0.885). CONCLUSION: The CECT-derived imaging features, including TNR, Thickness, tumor margin, and their combination, can be used as predictors of LVI status for patients with ESCC.

Gestational hypertensive disease and small for gestational age infants in twin pregnancy: A systematic review and meta-analysis.

AIM: The review is to explore the connection between gestational hypertension diseases (GHD) and small for gestational age (SGA) in twin pregnancies. METHODS: According to the recommendations of PRISMA, relevant studies were systematically searched through PubMed, Web of Science, Cochrane Library, Embase from inception until January 16, 2022. Subgroup analysis was performed according to chorionicity and diagnostic criteria of SGA. Odds ratios (OR) were assessed to judge the link between GHD and SGA in twin pregnant women. A random-effect model was used to estimate the pooled hazard ratio when there was significant heterogeneity (I2  > 50%); otherwise, a fixed-effect model was conducted. RESULTS: Seven articles containing 470 589 twin pregnant women were included. The increased risk of SGA was connected to the twin pregnancies complicated with GHD (OR = 1.57, 95% confidence interval [CI] = 1.10-2.24, p = 0.01). After subgroup analysis, the connection between SGA and GHD had no statistical significance (OR = 1.17, 95% CI = 0.95-1.44, p = 0.14) when the enrolled studies using the SGA diagnosis referred to singleton birth weight, but significant (OR = 2.14, 95% CI = 1.77-2.60, p<0.001) in the group using the SGA diagnosis referred to twin birth weight. Stratified by chorionicity, SGA was relevant to GHD in the dichorionic (DC) group (OR = 1.68, 95% CI = 1.17-2.42, p = 0.005), while not in the monochorionic (MC) group (OR = 1.68, 95% CI = 0.93-3.03, p = 0.09). More future articles are warranted to confirm these outcomes. CONCLUSIONS: Our review demonstrated that GHD in DC twin pregnancies was related to an enlarged risk of SGA. Two SGA diagnosis references led to different results. Twin pregnancies complicated with GHD were at significantly higher risk of SGA when twin birth weight reference was used.

The stem rust fungus Puccinia graminis f. sp. tritici induces centromeric small RNAs during late infection that are associated with genome-wide DNA methylation.

BACKGROUND: Silencing of transposable elements (TEs) is essential for maintaining genome stability. Plants use small RNAs (sRNAs) to direct DNA methylation to TEs (RNA-directed DNA methylation; RdDM). Similar mechanisms of epigenetic silencing in the fungal kingdom have remained elusive. RESULTS: We use sRNA sequencing and methylation data to gain insight into epigenetics in the dikaryotic fungus Puccinia graminis f. sp. tritici (Pgt), which causes the devastating stem rust disease on wheat. We use Hi-C data to define the Pgt centromeres and show that they are repeat-rich regions (~250 kb) that are highly diverse in sequence between haplotypes and, like in plants, are enriched for young TEs. DNA cytosine methylation is particularly active at centromeres but also associated with genome-wide control of young TE insertions. Strikingly, over 90% of Pgt sRNAs and several RNAi genes are differentially expressed during infection. Pgt induces waves of functionally diversified sRNAs during infection. The early wave sRNAs are predominantly 21 nts with a 5' uracil derived from genes. In contrast, the late wave sRNAs are mainly 22-nt sRNAs with a 5' adenine and are strongly induced from centromeric regions. TEs that overlap with late wave sRNAs are more likely to be methylated, both inside and outside the centromeres, and methylated TEs exhibit a silencing effect on nearby genes. CONCLUSIONS: We conclude that rust fungi use an epigenetic silencing pathway that might have similarity with RdDM in plants. The Pgt RNAi machinery and sRNAs are under tight temporal control throughout infection and might ensure genome stability during sporulation.

LSO-FastSLAM: A New Algorithm to Improve the Accuracy of Localization and Mapping for Rescue Robots.

This paper improves the accuracy of a mine robot's positioning and mapping for rapid rescue. Specifically, we improved the FastSLAM algorithm inspired by the lion swarm optimization method. Through the division of labor between different individuals in the lion swarm optimization algorithm, the optimized particle set distribution after importance sampling in the FastSLAM algorithm is realized. The particles are distributed in a high likelihood area, thereby solving the problem of particle weight degradation. Meanwhile, the diversity of particles is increased since the foraging methods between individuals in the lion swarm algorithm are different so that improving the accuracy of the robot's positioning and mapping. The experimental results confirmed the improvement of the algorithm and the accuracy of the robot.

Silencing of transcription factor encoding gene StTCP23 by small RNAs derived from the virulence modulating region of potato spindle tuber viroid is associated with symptom development in potato.

Viroids are small, non-protein-coding RNAs which can induce disease symptoms in a variety of plant species. Potato (Solanum tuberosum L.) is the natural host of Potato spindle tuber viroid (PSTVd) where infection results in stunting, distortion of leaves and tubers and yield loss. Replication of PSTVd is accompanied by the accumulation of viroid-derived small RNAs (sRNAs) proposed to play a central role in disease symptom development. Here we report that PSTVd sRNAs direct RNA silencing in potato against StTCP23, a member of the TCP (teosinte branched1/Cycloidea/Proliferating cell factor) transcription factor family genes that play an important role in plant growth and development as well as hormonal regulation, especially in responses to gibberellic acid (GA). The StTCP23 transcript has 21-nucleotide sequence complementarity in its 3' untranslated region with the virulence-modulating region (VMR) of PSTVd strain RG1, and was downregulated in PSTVd-infected potato plants. Analysis using 3' RNA ligase-mediated rapid amplification of cDNA ends (3' RLM RACE) confirmed cleavage of StTCP23 transcript at the expected sites within the complementarity with VMR-derived sRNAs. Expression of these VMR sRNA sequences as artificial miRNAs (amiRNAs) in transgenic potato plants resulted in phenotypes reminiscent of PSTVd-RG1-infected plants. Furthermore, the severity of the phenotypes displayed was correlated with the level of amiRNA accumulation and the degree of amiRNA-directed down-regulation of StTCP23. In addition, virus-induced gene silencing (VIGS) of StTCP23 in potato also resulted in PSTVd-like phenotypes. Consistent with the function of TCP family genes, amiRNA lines in which StTCP23 expression was silenced showed a decrease in GA levels as well as alterations to the expression of GA biosynthesis and signaling genes previously implicated in tuber development. Application of GA to the amiRNA plants minimized the PSTVd-like phenotypes. Taken together, our results indicate that sRNAs derived from the VMR of PSTVd-RG1 direct silencing of StTCP23 expression, thereby disrupting the signaling pathways regulating GA metabolism and leading to plant stunting and formation of small and spindle-shaped tubers.

Tomato yellow leaf curl virus intergenic siRNAs target a host long noncoding RNA to modulate disease symptoms.

Tomato yellow leaf curl virus (TYLCV) and its related begomoviruses cause fast-spreading diseases in tomato worldwide. How this virus induces diseases remains largely unclear. Here we report a noncoding RNA-mediated model to elucidate the molecular mechanisms of TYLCV-tomato interaction and disease development. The circular ssDNA genome of TYLCV contains a noncoding intergenic region (IR), which is known to mediate viral DNA replication and transcription in host cells, but has not been reported to contribute directly to viral disease development. We demonstrate that the IR is transcribed in dual orientations during plant infection and confers abnormal phenotypes in tomato independently of protein-coding regions of the viral genome. We show that the IR sequence has a 25-nt segment that is almost perfectly complementary to a long noncoding RNA (lncRNA, designated as SlLNR1) in TYLCV-susceptible tomato cultivars but not in resistant cultivars which contains a 14-nt deletion in the 25-nt region. Consequently, we show that viral small-interfering RNAs (vsRNAs) derived from the 25-nt IR sequence induces silencing of SlLNR1 in susceptible tomato plants but not resistant plants, and this SlLNR1 downregulation is associated with stunted and curled leaf phenotypes reminiscent of TYLCV symptoms. These results suggest that the lncRNA interacts with the IR-derived vsRNAs to control disease development during TYLCV infection. Consistent with its possible function in virus disease development, over-expression of SlLNR1 in tomato reduces the accumulation of TYLCV. Furthermore, gene silencing of the SlLNR1 in the tomato plants induced TYLCV-like leaf phenotypes without viral infection. Our results uncover a previously unknown interaction between vsRNAs and host lncRNA, and provide a plausible model for TYLCV-induced diseases and host antiviral immunity, which would help to develop effective strategies for the control of this important viral pathogen.

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.

Mine MIMO Depth Receiver: An Intelligent Receiving Model Based on Densely Connected Convolutional Networks.

Multiple-input multiple-output (MIMO) systems suffer from high BER in the mining environment. In this paper, the mine MIMO depth receiver model is proposed. The model uses densely connected convolutional networks for feature extraction and constructs multiple binary classifiers to recover the original information. Compared with conventional MIMO receivers, the model has no error accumulation caused by processes such as decoding and demodulation. The experimental results show that the model has better performance than conventional decoding methods under different modulation codes and variations in the number of transmitting terminals. Furthermore, we demonstrate that the model can still achieve effective decoding and recover the original information with some data loss at the receiver.

Identifying potential metastasis-related long non-coding RNAs, microRNAs, and message RNAs in the esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the predominant form with the highest incidence. We aimed to find metastasis-related differentially expressed long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and messenger RNA (mRNAs) in ESCC. We first obtained the lncRNAs, miRNAs, and mRNAs profiles. The differentially expressed lncRNAs, miRNAs, and mRNAs were obtained, followed by the functional annotation. Then the interaction networks of miRNA-mRNA, lncRNA-mRNA coexpression, lncRNA-miRNA, and lncRNA-miRNA-mRNA were constructed. In addition, systematic expression pattern analysis of differentially expressed lncRNAs, miRNA, and mRNA in the normal, metastasis, and nonmetastasis was performed. Survivability of differentially expressed lncRNAs, miRNAs, and mRNA was analyzed. A total of 613 differentially expressed lncRNAs, 35 differentially expressed miRNAs, and 1586 differentially expressed mRNAs were obtained. Several interactions of H19-hsa-mir-222-chromobox 2 (CBX2), H19-hsa-mir-330-phosphoinositide-3-kinase regulatory subunit 4 (PIK3R4), KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1)/CTB-89H12.4-hsa-mir-374a-vascular endothelial growth factor A (VEGFA), MALAT1/X inactive specific transcript (XIST)/XIST antisense RNA (TSIX)-hsa-mir-340-tumor necrosis factor receptor superfamily member 10A (NFRSF10A) were identified to play key roles in the metastasis of ESCC. In addition, KCNQ1OT1, TSIX, and XIST were significantly associated with the survival time of patients. In conclusion, our study may be helpful in understanding the pathological mechanism and providing new diagnostic and therapeutic biomarkers for ESCC.

Identification of Essential Sensitive Regions of the Aerolysin Nanopore for Single Oligonucleotide Analysis.

The aerolysin nanopore channel is one of the confined spaces for single molecule analysis which displays high spatial and temporal resolution for the discrimination of single nucleotides, identification of DNA base modification, and analyzing the structural transition of DNAs. However, to overcome the challenge of achieving the ultimate goal of the widespread real analytical application, it is urgent to probe the sensing regions of the aerolysin to further improve the sensitivity. In this paper, we explore the sensing regions of the aerolysin nanopore by a series of well-designed mutant nanopore experiments combined with molecular dynamics simulations-based electrostatic analysis. The positively charged lumen-exposed Lys-238, identified as one of the key sensing sites due to the presence of a deep valley in the electrostatic potentials, was replaced by different charged and sized amino acids. The results show that the translocation time of oligonucleotides through the nanopore can be readily modulated by the choice of the target amino acid at the 238 site. In particular, a 7-fold slower translocation at a voltage bias of +120 mV is observed with respect to the wild-type aerolysin, which provides a high resolution for methylated cytosine discrimination. We further determine that both the electrostatic properties and geometrical structure of the aerolysin nanopore are crucial to its sensing ability. These insights open ways for rationally designing the sensing mechanism of the aerolysin nanopore, thus providing a novel paradigm for nanopore sensing.

Extensive genetic and DNA methylation variation contribute to heterosis in triploid loquat hybrids.

We aim to overcome the unclear origin of the loquat and elucidate the heterosis mechanism of the triploid loquat. Here we investigated the genetic and epigenetic variations between the triploid plant and its parental lines using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified fragment length polymorphism (MSAP) analyses. We show that in addition to genetic variations, extensive DNA methylation variation occurred during the formation process of triploid loquat, with the triploid hybrid having increased DNA methylation compared to the parents. Furthermore, a correlation existed between genetic variation and DNA methylation remodeling, suggesting that genome instability may lead to DNA methylation variation or vice versa. Sequence analysis of the MSAP bands revealed that over 53% of them overlap with protein-coding genes, which may indicate a functional role of the differential DNA methylation in gene regulation and hence heterosis phenotypes. Consistent with this, the genetic and epigenetic alterations were associated closely to the heterosis phenotypes of triploid loquat, and this association varied for different traits. Our results suggested that the formation of triploid is accompanied by extensive genetic and DNA methylation variation, and these changes contribute to the heterosis phenotypes of the triploid loquats from the two cross lines.

Nicotiana small RNA sequences support a host genome origin of cucumber mosaic virus satellite RNA.

Satellite RNAs (satRNAs) are small noncoding subviral RNA pathogens in plants that depend on helper viruses for replication and spread. Despite many decades of research, the origin of satRNAs remains unknown. In this study we show that a ß-glucuronidase (GUS) transgene fused with a Cucumber mosaic virus (CMV) Y satellite RNA (Y-Sat) sequence (35S-GUS:Sat) was transcriptionally repressed in N. tabacum in comparison to a 35S-GUS transgene that did not contain the Y-Sat sequence. This repression was not due to DNA methylation at the 35S promoter, but was associated with specific DNA methylation at the Y-Sat sequence. Both northern blot hybridization and small RNA deep sequencing detected 24-nt siRNAs in wild-type Nicotiana plants with sequence homology to Y-Sat, suggesting that the N. tabacum genome contains Y-Sat-like sequences that give rise to 24-nt sRNAs capable of guiding RNA-directed DNA methylation (RdDM) to the Y-Sat sequence in the 35S-GUS:Sat transgene. Consistent with this, Southern blot hybridization detected multiple DNA bands in Nicotiana plants that had sequence homology to Y-Sat, suggesting that Y-Sat-like sequences exist in the Nicotiana genome as repetitive DNA, a DNA feature associated with 24-nt sRNAs. Our results point to a host genome origin for CMV satRNAs, and suggest novel approach of using small RNA sequences for finding the origin of other satRNAs.

Genetic enhancement of oil content in potato tuber (Solanum tuberosum L.) through an integrated metabolic engineering strategy.

Potato tuber is a high yielding food crop known for its high levels of starch accumulation but only negligible levels of triacylglycerol (TAG). In this study, we evaluated the potential for lipid production in potato tubers by simultaneously introducing three transgenes, including WRINKLED 1 (WRI1), DIACYLGLYCEROL ACYLTRANSFERASE 1 (DGAT1) and OLEOSIN under the transcriptional control of tuber-specific (patatin) and constitutive (CaMV-35S) promoters. This coordinated metabolic engineering approach resulted in over a 100-fold increase in TAG accumulation to levels up to 3.3% of tuber dry weight (DW). Phospholipids and galactolipids were also found to be significantly increased in the potato tuber. The increase of lipids in these transgenic tubers was accompanied by a significant reduction in starch content and an increase in soluble sugars. Microscopic examination revealed that starch granules in the transgenic tubers had more irregular shapes and surface indentations when compared with the relatively smooth surfaces of wild-type starch granules. Ultrastructural examination of lipid droplets showed their close proximity to endoplasmic reticulum and mitochondria, which may indicate a dynamic interaction with these organelles during the processes of lipid biosynthesis and turnover. Increases in lipid levels were also observed in the transgenic potato leaves, likely due to the constitutive expression of DGAT1 and incomplete tuber specificity of the patatin promoter. This study represents an important proof-of-concept demonstration of oil increase in tubers and provides a model system to further study carbon reallocation during development of nonphotosynthetic underground storage organs.

Incorporating Transgenerational Epigenetic Inheritance into Ecological Risk Assessment Frameworks.

Chronic exposure to environmental contaminants can induce heritable "transgenerational" modifications to organisms, potentially affecting future ecosystem health and functionality. Incorporating transgenerational epigenetic heritability into risk assessment procedures has been previously suggested. However, a critical review of existing literature yielded numerous studies claiming transgenerational impacts, with little compelling evidence. Therefore, contaminant-induced epigenetic inheritance may be less common than is reported in the literature. We identified a need for multigeneration epigenetic studies that extend beyond what could be deemed "direct exposure" to F1 and F2 gametes and also include subsequent multiple nonexposed generations to adequately evaluate transgenerational recovery times. Also, increased experimental replication is required to account for the highly variable nature of epigenetic responses and apparent irreproducibility of current studies. Further, epigenetic end points need to be correlated with observable detrimental organism changes before a need for risk management can be properly determined. We suggest that epigenetic-based contaminant studies include concentrations lower than current "EC10-20" or "Lowest Observable Effect Concentrations" for the organism's most sensitive phenotypic end point, as higher concentrations are likely already regulated. Finally, we propose a regulatory framework and optimal experimental design that enables transgenerational epigenetic effects to be assessed and incorporated into conventional ecotoxicological testing.

Epigenetic Mechanisms: An Emerging Player in Plant-Microbe Interactions.

Plants have developed diverse molecular and cellular mechanisms to cope with a lifetime of exposure to a variety of pathogens. Host transcriptional reprogramming is a central part of plant defense upon pathogen recognition. Recent studies link DNA methylation and demethylation as well as chromatin remodeling by posttranslational histone modifications, including acetylation, methylation, and ubiquitination, to changes in the expression levels of defense genes upon pathogen challenge. Remarkably these inducible defense mechanisms can be primed prior to pathogen attack by epigenetic modifications and this heightened resistance state can be transmitted to subsequent generations by inheritance of these modification patterns. Beside the plant host, epigenetic mechanisms have also been implicated in virulence development of pathogens. This review highlights recent findings and insights into epigenetic mechanisms associated with interactions between plants and pathogens, in particular bacterial and fungal pathogens, and demonstrates the positive role they can have in promoting plant defense.

Ubiquitous miR159 repression of MYB33/65 in Arabidopsis rosettes is robust and is not perturbed by a wide range of stresses.

BACKGROUND: The microR159 (miR159) - GAMYB pathway is conserved in higher plants, where GAMYB, expression promotes programmed cell death in seeds (aleurone) and anthers (tapetum). In cereals, restriction of GAMYB expression to seeds and anthers is mainly achieved transcriptionally, whereas in Arabidopsis this is achieved post-transcriptionally, as miR159 silences GAMYB (MYB33 and MYB65) in vegetative tissues, but not in seeds and anthers. However, we cannot rule out a role for miR159-MYB33/65 pathway in Arabidopsis vegetative tissues; a loss-of-function mir159 Arabidopsis mutant displays strong pleiotropic defects and numerous reports have documented changes in miR159 abundance during stress and hormone treatments. Hence, we have investigated the functional role of this pathway in vegetative tissues. RESULTS: It was found that the miR159-MYB33/65 pathway was ubiquitously present throughout rosette development. However, miR159 appears to continuously repress MYB33/MYB65 expression to levels that have no major impact on rosette development. Inducible inhibition of miR159 resulted in MYB33/65 de-repression and associated phenotypic defects, indicating that a potential role in vegetative development is only possible through MYB33 and MYB65 if miR159 levels decrease. However, miR159 silencing of MYB33/65 appeared extremely robust; no tested abiotic stress resulted in strong miR159 repression. Consistent with this, the stress responses of an Arabidopsis mutant lacking the miR159-MYB33/65 pathway were indistinguishable from wild-type. Moreover, expression of viral silencing suppressors, either via transgenesis or viral infection, was unable to prevent miR159 repression of MYB33/65, highlighting the robustness of miR159-mediated silencing. CONCLUSIONS: Despite being ubiquitously present, molecular, genetic and physiological analysis failed to find a major functional role for the miR159-MYB33/65 pathway in Arabidopsis rosette development or stress response. Although it is likely that this pathway is important for a stress not tested here or in different plant species, our findings argue against the miR159-MYB33/65 pathway playing a major conserved role in general stress response. Finally, in light of the robustness of miR159-mediated repression of MYB33/65, it appears unlikely that low fold-level changes of miR159 abundance in response to stress would have any major physiological impact in Arabidopsis.