CRISPR/Cas9-mediated multi-allelic gene targeting in sugarcane confers herbicide tolerance

Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2n = 100 – 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles via template-mediated and homology-directed repair (HDR) of DNA double strand breaks induced by the programmable nuclease CRISPR/Cas9. The evaluation of 146 independently transformed plants from five independent experiments revealed a targeted nucleotide replacement that resulted in both targeted amino acid substitutions W574L and S653I in the acetolactate synthase (ALS) in 11 lines in addition to single, targeted amino acid substitutions W574L or S653I in 25 or 18 lines, respectively. Co-editing of up to three ALS copies/alleles that confer herbicide tolerance was confirmed by Sanger sequencing of cloned long polymerase chain reaction (PCR) amplicons. This work will enable crop improvement by conversion of inferior alleles to superior alleles through targeted nucleotide substitutions

Expression Analysis of TaNAC69-1 and TtNAMB-2, Wheat NAC Family Transcription Factor Genes Under Abiotic Stress Conditions in Durum Wheat (Triticum turgidum)

NAC-type plant-specific transcription factor genes encode proteins that play important roles in abiotic stress responses, as well as regulation of plant development. In the current study, expression profiles of wheat NAC-type transcription factor genes, TaNAC69-1 and TtNAMB-2, were examined under drought, salt, cold, and heat stress conditions in wheat. Based on reverse transcription quantitative PCR results, TaNAC69-1 was strongly expressed under drought, salinity, and high-temperature stress conditions. Compared to control samples, a quick response at the transcription level of TaNAC69-1 was observed after 3 h of salt treatment with a ninefold upregulation. Highest level of expression was observed at 24 and 48 h posttreatment under heat and salinity treatments, respectively. Meanwhile, expression of TtNAMB-2 was significantly induced by salt and low-temperature stresses. Salt treatment induced expression of TtNAMB-2 and caused a 13-fold increase in transcript copy numbers at 48 h posttreatment. Examination of expression changes under abiotic stresses may provide important information for understanding roles of TaNAC69-1 and TtNAMB-2 genes which might be involved in response to environmental stresses

Transcriptome profiling of wheat under heat and cold stress treatments

Microarray analysis was performed to indicate effects of cold and heat stress treatments on global gene expression profiles of wheat. Two groups were generated for comparison of microarray data analysis. Cold and heat conditions were separately compared to control condition. The Affymetrix GeneChip® Wheat Genome Array contains 61,127 probe sets designed to target 55,052 wheat transcripts. Out of these more than 60,000 probes, 12,895 (21.5%) were found to be differentially expressed at least p value lower than 0.05 and fold change (FC) higher than 2. Alternation in expression level of about 2550 probe sets was common after the cold and heat stress treatments. Approximately 3600 and 5500 probe sets were differentially expressed after the heat and cold stresses, respectively. Differentially regulated genes show that temperature changes directly affected a large and complex transcriptional network associated with defense, metabolism and development. Genes involved in cold stress-responsive and different cold acclimation proteins were extremely up-regulated upon exposure to cold stress. Both expression levels of small and large sub-unit heat shock proteins significantly increased following heat stress period

Physiological, Biochemical, and Transcriptomic Responses to Boron Toxicity in Leaf and Root Tissues of Contrasting Wheat Cultivars

In this study, we examined physiological, biochemical, and transcriptomic responses to toxic boron (B) treatment in leaves and roots of two wheat cultivars in order to gain better insight into B response or tolerance mechanisms. Delayed development and reduced vigor caused by high B were not observed in leaves and roots of both cultivars. Length, wet weight, and dry weight were not markedly changed under B toxicity. In leaves, when compared to control, 995 and 892 genes were significantly expressed at least twofold under B toxicity in Atay and Bolal, respectively. In roots, expressions of 1248 and 957 genes were responsive to B toxicity in Atay and Bolal, respectively. In leaf and root tissues, B toxicity induced more genes related to protein degradation in Atay than those in Bolal. These differences in transcriptome were attributed to higher B accumulation in the sensitive cultivar which required high level of metabolic adjustment. B toxicity stress did not cause any significant change in photosynthetic activity and contents of proline and glycine betaine in both cultivars. Coordinately, we did not find any differentially expressed genes required for proline and glycine betaine metabolisms. Genes related to hormone signaling, kinases, transcription factors such as WRKY and MYB, and key enzymes in reactive oxygen species (ROS) scavenging mechanisms were differentially affected by B toxicity in both cultivars. Among commonly regulated genes in Atay and Bolal, glutathione S-transferase (GST) and NIP4;1 (nodulin-26-like intrinsic proteins) genes stand out as prominent actors in B stress response

Związek między niskimi stężeniami bilirubiny a zjawiskiem zwolnionego przepływu wieńcowego

Background and aim: Increasing evidence suggests an inverse relationship between bilirubin levels and cardiovascular disease. The present study evaluated the effect of bilirubin level on the slow coronary flow (SCF) phenomenon. Methods: This study was cross-sectional and observational. We enrolled 222 consecutive patients who underwent coronary angiography for suspected ischaemic heart disease and were found to have normal or near-normal coronary arteries. Then, bilirubin levels were measured and coronary flow rate was assessed using the thrombolysis in myocardial infarction (TIMI) frame count. SCF was defined as a TIMI frame count > 27 frames. Results: SCF was observed in at least one coronary vessel in 22 of the 222 subjects, indicating a prevalence of 10%. Serum bilirubin levels were significantly decreased in the SCF group. In multivariate logistic regression analysis, total bilirubin and diabetes mellitus were independent risk factors for SCF. Furthermore, after adjusting for age, sex, and cardiovascular disease risk factors, serum bilirubin level (B = –0.34, p < 0.001) was independently associated with TIMI frame count. Conclusions: These findings suggest that serum total bilirubin levels may be a useful marker for patients with the SCF phenomenon. We believe that further studies are needed to clarify the role of bilirubin in patients with SCF.Wstęp i cel: Coraz większa liczba dowodów naukowych wskazuje, że istnieje odwrotna zależność między stężeniem bilirubiny a chorobami sercowo-naczyniowymi. W niniejszym badaniu oceniono wpływ stężenia bilirubiny na zjawisko zwolnionego przepływu wieńcowego (SCF). Metody: Do tego przekrojowego badania obserwacyjnego włączono 222 kolejnych pacjentów, u których wykonano koronarografię z powodu podejrzenia choroby niedokrwiennej serca i stwierdzono prawidłowe lub prawie prawidłowe tętnice wieńcowe. Następnie zmierzono stężenie bilirubiny i oceniono szybkość przepływu wieńcowego, stosując metodę określania liczby klatek do pojawienia się środka cieniującego, użytą w badaniu Thrombolysis in Myocardial Infarction (TIMI). Zwolniony przepływ wieńcowy zdefiniowano jako liczbę klatek TIMI wynoszącą > 27. Wyniki: W badanej populacji SCF w co najmniej 1 tętnicy wieńcowej stwierdzono u 22 chorych, co oznacza chorobowość wynoszącą 10%. Stężenia bilirubiny w surowicy były istotnie mniejsze w grupie osób z SCF. W wieloczynnikowej analizie regresji logistycznej całkowite stężenie bilirubiny i cukrzyca stanowiły niezależne czynniki ryzyka SCF. Ponadto po skorygowaniu względem wieku, płci i czynników ryzyka chorób sercowo-naczyniowych stężenie bilirubiny w surowicy (B = –0,34; p < 0,001) było niezależnie związane z liczbą klatek TIMI. Wnioski: Podsumowując, powyższe rezultaty wskazują, że całkowite stężenie bilirubiny w surowicy może być użytecznym wskaźnikiem u chorych, u których występuje SCF. Autorzy uważają, że należy przeprowadzić dalsze badania w celu sprecyzowania znaczenia oznaczeń stężenia bilirubiny u chorych z SCF

Low bilirubin levels are associated with coronary slow flow phenomenon

Background and aim: Increasing evidence suggests an inverse relationship between bilirubin levels and cardiovascular disease. The present study evaluated the effect of bilirubin level on the slow coronary flow (SCF) phenomenon