Seed yield and oil content are two important agricultural characteristics in oil crop breeding, and a lot of functional gene research is being concentrated on increasing these factors. In this study, by differential gene expression analyses between rapeseed lines (zy036 and 51070) which exhibit different levels of seed oil production, BnGRF2 ( Brassica napus growth-regulating factor 2-like gene) was identified in the high oil-producing line zy036. To elucidate the possible roles of BnGRF2 in seed oil production, the cDNA sequences of the rapeseed GRF2 gene were isolated. The Blastn result showed that rapeseed contained BnGRF2a/2b which were located in the A genome (A1 and A3) and C genome (C1 and C6), respectively, and the dominantly expressed gene BnGRF2a was chosen for transgenic research. Analysis of 35S- BnGRF2a transgenic Arabidopsis showed that overexpressed BnGRF2a resulted in an increase in seed oil production of >50%. Moreover, BnGRF2a also induced a >20% enlargement in extended leaves and >40% improvement in photosynthetic efficiency because of an increase in the chlorophyll content. Furthermore, transcriptome analyses indicated that some genes associated with cell proliferation, photosynthesis, and oil synthesis were up-regulated, which revealed that cell number and plant photosynthesis contributed to the increased seed weight and oil content. Because of less efficient self-fertilization induced by the longer pistil in the 35S- BnGRF2a transgenic line, Napin- BnGRF2a transgenic lines were further used to identify the function of BnGRF2 , and the results showed that seed oil production also could increase >40% compared with the wild-type control. The results suggest that improvement to economically important characteristics in oil crops may be achieved by manipulation of the GRF2 expression level.

Pharmacological, laser scanning confocal microscopic (LSCM), real-time PCR and spectrophotographic approaches are used to study the roles of hydrogen sulfide (H 2 S) and nitric oxide (NO) in signaling transduction of stomatal movement response to ethylene in Arabidopsis thaliana. In the present study, inhibitors of H 2 S synthesis were found to block ethylene-induced stomatal closure of Arabidopsis. Treatment with ethylene induced H 2 S generation and increased L-/D-cysteine desulfhydrase (pyridoxalphosphate-dependent enzyme) activity in leaves. Quantitative PCR analysis showed AtL-CDes and AtD-CDes transcripts were induced by ethylene. It is suggested that ethylene-induced H 2 S levels and L-/D-cysteine desulfhydrase activity decreased when NO was compromised. The data clearly show that ethylene was able to induce H 2 S generation and stomatal closure in Atnoa1 plants, but failed in the Atnia1,nia2 mutant. Inhibitors of H 2 S synthesis had no effect on ethylene-induced NO accumulation and nitrate reductase (NR) activity in guard cells or leaves of Arabidopsis, whereas ethylene was able to induce NO synthesis. Therefore, we conclude that H 2 S and NO are involved in the signal transduction pathway of ethylene-induced stomatal closure. In Arabidopsis, H 2 S may represent a novel downstream indicator of NO during ethylene-induced stomatal movement. hydrogen sulfide, nitric oxide, L-/D-cysteine desulfhydrase, Arabidopsis thaliana, ethylene, stomatal closure Citation: Liu J, Hou L X, Liu G H, et al. Hydrogen sulfide induced by nitric oxide mediates ethylene-induced stomatal closure of Arabidopsis thaliana.

Hydrogen sulfide (H2 S) is a newly-discovered signaling molecule in plants and has caused increasing attention in recent years, but its function in stomatal movement is unclear. In plants, H2 S is synthesized via cysteine degradation catalyzed by D-/L-cysteine desulfhydrase (D-/L-CDes). AtD-/L-CDes::GUS transgenic Arabidopsis thaliana (L.) Heynh. plants were generated and used to investigate gene expression patterns, and results showed that AtD-/L-CDes can be expressed in guard cells. We also determined the subcellular localization of AtD-/L-CDes using transgenic plants of AtD-/L-CDes::GFP, and the results showed that AtD-CDes and AtL-CDes are located in the chloroplast and in the cytoplasm, respectively. The transcript levels of AtD-CDes and AtL-CDes were affected by the chemicals that cause stomatal closure. Among these factors, ACC, a precursor of ethylene, has the most significant effect, which indicates that the H2 S generated from D-/L-CDes may play an important role in ethylene-induced stomatal closure. Meanwhile, H2 S synthetic inhibitors significantly inhibited ethylene-induced stomatal closure in Arabidopsis. Ethylene treatment caused an increase of H2 S production and of AtD-/L-CDes activity in Arabidopsis leaves. AtD-/L-CDes over-expressing plants exhibited enhanced induction of stomatal closure compared to the wild-type after ethylene treatment; however, the effect was not observed in the Atd-cdes and Atl-cdes mutants. In conclusion, our results suggest that the D-/L-CDes-generated H2 S is involved in the regulation of ethylene-induced stomatal closure in Arabidopsis thaliana.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.

Induction of a pluripotent cell mass termed callus is the first step in an in vitro plant regeneration system, which is required for subsequent regeneration of new organs or whole plants. However, the early molecular mechanism underlying callus initiation is largely elusive. Here, we analyzed the dynamic transcriptome profiling of callus initiation in Arabidopsis aerial and root explants and identified 1342 differentially expressed genes in both explants after incubation on callus-inducing medium. Detailed categorization revealed that the differentially expressed genes were mainly related to hormone homeostasis and signaling, transcriptional and post transcriptional regulations, protein phosphorelay cascades and DNA- or chromatin-modification. Further characterization showed that overexpression of two transcription factors, HB52 or CRF3, resulted in the callus formation in transgenic plants without exogenous auxin. Therefore, our comprehensive analyses provide some insight into the early molecular regulations during callus initiation and are useful for further identification of the regulators governing callus formation.

SUMMARYCell proliferation is integrated into developmental progression in multicellular organisms, including plants, and the regulation of cell division is of pivotal importance for plant growth and development. Here, we report the identification of an Arabidopsis SMALL ORGAN 2 (SMO2) gene that functions in regulation of the progression of cell division during organ growth. The smo2 knockout mutant displays reduced size of aerial organs and shortened roots, due to the decreased number of cells in these organs. Further analyses reveal that disruption of SMO2 does not alter the developmental timing but reduces the rate of cell production during leaf and root growth. Moreover, smo2 plants exhibit a constitutive activation of cell cycle-related genes and overaccumulation of cells expressing CYCB1;1:b-glucuronidase (CYCB1;1:GUS) during organogenesis, suggesting that smo2 has a defect in G 2 -M phase progression in the cell cycle. SMO2 encodes a functional homologue of yeast TRM112, a plurifunctional component involved in a few cellular events, including tRNA and protein methylation. In addition, the mutation of SMO2 does not appear to affect endoreduplication in Arabidopsis leaf cells. Taken together we postulate that Arabidopsis SMO2 is a conserved yeast TRM112 homologue and SMO2-mediated cellular events are required for proper progression of cell division in plant growth and development.

Rare genetic diseases account for a considerable amount of fatalities and even their 'mild' or 'non-lethal' forms can produce drastic and undesirable discomfort to affected individuals. Various gene therapeutic approaches were tested for developing novel therapeutic concepts to treat these genetic diseases. Sleeping Beauty (SB) transposase represents one of these gene therapeutic systems which can be utilized for stable phenotypic correction. It is a transposable element which was resurrected and optimized for transposing genetic elements resulting in somatic integration of the transgene. Because of its versatile activity in many different organs, SB transposase has been explored for ex-vivo gene delivery and in vivo gene delivery including recently launched clinical trials based on engineered T-cells for tumor therapy and approaches to treat retinal degenerations. Here we will provide a state-of-the-art overview of preclinical studies for treatment of rare genetic diseases based on the SB transposase system for stable correction of the genetic defect. In this review, diseases affecting the blood system, the connective tissue, the immune system, the metabolism, and the nervous system and their treatment utilizing the SB transposase system will be discussed. Moreover, advantages and disadvantages of SB transposase-based gene therapeutic approaches will be mentioned. Although improvements of the SB transposase systems regarding genotoxicity and efficient delivery especially for applications in large mammals are desirable, the SB transposase system remains to hold great promise for curing rare genetic disease.

We investigated changes in cytosolic pH and nitric oxide (NO) during ethylene-induced stomatal closure in Arabidopsis thaliana using pharmacological, laser scanning confocal microscopy (LSCM), and spectrophotography techniques. Treatment with ethephon (a direct source of ethylene when applied to plants) and 1-aminocycloaminopropane-1-carboxylic acid (ACC, an ethylene precursor) resulted in a rapid accumulation of NO and cytosolic alkalinization in guard cells. Acetic acid (a weak acid) and sodium orthovanadate (NaVO 3 ; a plasmalemma H + -ATPase inhibitor) reduced stomatal closure induced by ethylene and blocked ethylene-induced activity of nitrate reductase. However, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a NO scavenger, had no effect. These results suggest that NO production is downstream of the rise in cytosolic pH in A. thaliana. Arabidopsis thaliana, ethylene, nitric oxide, pH, stomatal movement Citation:Liu J, Liu G H, Hou L X, et al. Ethylene-induced nitric oxide production and stomatal closure in Arabidopsis thaliana depending on changes in cytosolic pH.