Heterologous Expression of ATG8c from Soybean Confers Tolerance to Nitrogen Deficiency and Increases Yield in Arabidopsis

Nitrogen is an essential element for plant growth and yield. Improving Nitrogen Use Efficiency (NUE) of crops could potentially reduce the application of chemical fertilizer and alleviate environmental damage. To identify new NUE genes is therefore an important task in molecular breeding. Macroautophagy (autophagy) is an intracellular process in which damaged or obsolete cytoplasmic components are encapsulated in double membraned vesicles termed autophagosomes, then delivered to the vacuole for degradation and nutrient recycling. One of the core components of autophagosome formation, ATG8, has been shown to directly mediate autophagosome expansion, and the transcript of which is highly inducible upon starvation. Therefore, we postulated that certain homologs of Saccharomyces cerevisiae ATG8 (ScATG8) from crop species could have potential for NUE crop breeding. A soybean (Glycine max, cv. Zhonghuang-13) ATG8, GmATG8c, was selected from the 11 family members based on transcript analysis upon nitrogen deprivation. GmATG8c could partially complement the yeast atg8 mutant. Constitutive expression of GmATG8c in soybean callus cells not only enhanced nitrogen starvation tolerance of the cells but accelerated the growth of the calli. Transgenic Arabidopsis over-expressing GmATG8c performed better under extended nitrogen and carbon starvation conditions. Meanwhile, under optimum growth conditions, the transgenic plants grew faster, bolted earlier, produced larger primary and axillary inflorescences, eventually produced more seeds than the wild-type. In average, the yield was improved by 12.9%. We conclude that GmATG8c may serve as an excellent candidate for breeding crops with enhanced NUE and better yield

Doing synthetic biology with photosynthetic microorganisms

The use of photosynthetic microbes as synthetic biology hosts for the sustainable production of commodity chemicals and even fuels has received increasing attention over the last decade. The number of studies published, tools implemented, and resources made available for microalgae have increased beyond expectations during the last few years. However, the tools available for genetic engineering in these organisms still lag those available for the more commonly used heterotrophic host organisms. In this mini-review, we provide an overview of the photosynthetic microbes most commonly used in synthetic biology studies, namely cyanobacteria, chlorophytes, eustigmatophytes and diatoms. We provide basic information on the techniques and tools available for each model group of organisms, we outline the state-of-the-art, and we list the synthetic biology tools that have been successfully used. We specifically focus on the latest CRISPR developments, as we believe that precision editing and advanced genetic engineering tools will be pivotal to the advancement of the field. Finally, we discuss the relative strengths and weaknesses of each group of organisms and examine the challenges that need to be overcome to achieve their synthetic biology potential.Peer reviewe

RT-PCR analysis of ATG genes in Cucumis sativus during stress conditions

The expression of different splice variance of ATG genes in Cucumis sativus under stresses conditions including nitrogen starvation, oxidative stress, and pathogen infection were examined using realtime-PCR. The nitrogen starvation experiment was performed using five-day-old seedlings of C. sativus grown on liquid 1/2 MS medium with and without nitrogen for 2, 4 and 6 days (Oka et al., 2012). Oxidative stress was conducted using two-week-old seedlings grown on soil. These plants were sprayed once with 50 µM Methyl viologen (MV) prepared in 0.05% (v/v) Tween 20 (Song et al., 2005), and leaves samples were collected at 3, 6, 9, 12 and 15 days after the treatment. For pathogen responses, seven-day-old seedlings of C. sativus grown on soil were inoculated with sporangia of the fungi causing downy mildew disease (Pseudoperonospora cubensis). Samples were collected at 6, 12, 24, 72 and 168 h after inoculation. For each experiment, three replicates were collected for each time point before frozen in liquid nitrogen and stored at - 80 °CThe real-time PCR analysis was performed in duplicate assays in 96-well plates; each 10 µl reaction consisted of 2x QPCR Green Master Mix (Biotechrabbit, Germany), 5 µmol forward and reverse primers and 1 ng cDNA. The primers were designed to span the junction that differ between transcript variances. The data of Ct values obtained from two different experimental RNA samples were directly normalized to a housekeeping gene, CsActin. The fold-change in expression of the gene of interest between each timepoint was then calculated as 2^ (-ΔΔCt). Statistical differences in the level expression compared to the expression at time point 0 or that of ATG7 were analyzed with student’s T test with two-tailed distribution.THIS DATASET IS ARCHIVED AT DANS/EASY, BUT NOT ACCESSIBLE HERE. TO VIEW A LIST OF FILES AND ACCESS THE FILES IN THIS DATASET CLICK ON THE DOI-LINK ABOV

RT-PCR analysis of ATG genes and western blot analysis of ATG8 in Cucumis sativus during stress conditions

The expression of different splice variance of ATG genes in Cucumis sativus under stresses conditions including nitrogen starvation, oxidative stress, and pathogen infection were examined using realtime-PCR. The nitrogen starvation experiment was performed using five-day-old seedlings of C. sativus grown on liquid 1/2 MS medium with and without nitrogen for 2, 4 and 6 days (Oka et al., 2012). Oxidative stress was conducted using two-week-old seedlings grown on soil. These plants were sprayed once with 50 µM Methyl viologen (MV) prepared in 0.05% (v/v) Tween 20 (Song et al., 2005), and leaves samples were collected at 3, 6, 9, 12 and 15 days after the treatment. For pathogen responses, seven-day-old seedlings of C. sativus grown on soil were inoculated with sporangia of the fungi causing downy mildew disease (Pseudoperonospora cubensis). Samples were collected at 6, 12, 24, 72 and 168 h after inoculation. For each experiment, three replicates were collected for each time point before frozen in liquid nitrogen and stored at - 80 °CThe real-time PCR analysis was performed in duplicate assays in 96-well plates; each 10 µl reaction consisted of 2x QPCR Green Master Mix (Biotechrabbit, Germany), 5 µmol forward and reverse primers and 1 ng cDNA. The primers were designed to span the junction that differ between transcript variances. The data of Ct values obtained from two different experimental RNA samples were directly normalized to a housekeeping gene, CsActin. The fold-change in expression of the gene of interest between each timepoint was then calculated as 2^ (-ΔΔCt). Statistical differences in the level expression compared to the expression at time point 0 or that of ATG7 were analyzed with student’s T test with two-tailed distribution.Protein extraction and immunoblot analysisSamples were ground and resuspended in extraction buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 10% (v/v) glycerol, 0.5% (v/v) IGEPAL CA-360, 1 mM phenylmethylsulfonyl fluoride) on ice. Crude proteins were separated on18% SDS-PAGE before stained using Coomassie blue or transferred onto a PVDF membrane (GE Healthcare). Immunodetection of ATG8 was performed using anti-CrATG8 (Pérez-Pérez et al., 2010), with a working concentration of the primary antibody at 1:1000 in PBS containing 2.5% BSA. The Coomassie Brilliant Blue staining of the gel was used to estimate the amount of protein loaded.THIS DATASET IS ARCHIVED AT DANS/EASY, BUT NOT ACCESSIBLE HERE. TO VIEW A LIST OF FILES AND ACCESS THE FILES IN THIS DATASET CLICK ON THE DOI-LINK ABOV