Evaluation of Parameters Affecting Agrobacterium-Mediated Transient Gene Expression in Industrial Hemp (Cannabis sativa L.).

Industrial hemp Cannabis sativa L. is an economically important crop mostly grown for its fiber, oil, and seeds. Due to its increasing applications in the pharmaceutical industry and a lack of knowledge of gene functions in cannabinoid biosynthesis pathways, developing an efficient transformation platform for the genetic engineering of industrial hemp has become necessary to enable functional genomic and industrial application studies. A critical step in the development of Agrobacterium tumefaciens-mediated transformation in the hemp genus is the establishment of optimal conditions for T-DNA gene delivery into different explants from which whole plantlets can be regenerated. As a first step in the development of a successful Agrobacterium tumefaciens-mediated transformation method for hemp gene editing, the factors influencing the successful T-DNA integration and expression (as measured by transient ß-glucuronidase (GUS) and Green Florescent Protein (GFP) expression) were investigated. In this study, the parameters for an agroinfiltration system in hemp, which applies to the stable transformation method, were optimized. In the present study, we tested different explants, such as 1- to 3-week-old leaves, cotyledons, hypocotyls, root segments, nodal parts, and 2- to 3-week-old leaf-derived calli. We observed that the 3-week-old leaves were the best explant for transient gene expression. Fully expanded 2- to 3-week-old leaf explants, in combination with 30 min of immersion time, 60 µM silver nitrate, 0.5 µM calcium chloride, 150 µM natural phenolic compound acetosyringone, and a bacterial density of OD600nm = 0.4 resulted in the highest GUS and GFP expression. The improved method of genetic transformation established in the present study will be useful for the introduction of foreign genes of interest, using the latest technologies such as genome editing, and studying gene functions that regulate secondary metabolites in hemp.

Unraveling the genomic reorganization of polygalacturonase-inhibiting proteins in chickpea.

Polygalacturonase-inhibiting proteins (PGIPs) are cell wall proteins that inhibit pathogen polygalacturonases (PGs). PGIPs, like other defense-related proteins, contain extracellular leucine-rich repeats (eLRRs), which are required for pathogen PG recognition. The importance of these PGIPs in plant defense has been well documented. This study focuses on chickpea (Cicer arietinum) PGIPs (CaPGIPs) owing to the limited information available on this important crop. This study identified two novel CaPGIPs (CaPGIP3 and CaPGIP4) and computationally characterized all four CaPGIPs in the gene family, including the previously reported CaPGIP1 and CaPGIP2. The findings suggest that CaPGIP1, CaPGIP3, and CaPGIP4 proteins possess N-terminal signal peptides, ten LRRs, theoretical molecular mass, and isoelectric points comparable to other legume PGIPs. Phylogenetic analysis and multiple sequence alignment revealed that the CaPGIP1, CaPGIP3, and CaPGIP4 amino acid sequences are similar to the other PGIPs reported in legumes. In addition, several cis-acting elements that are typical of pathogen response, tissue-specific activity, hormone response, and abiotic stress-related are present in the promoters of CaPGIP1, CaPGIP3, and CaPGIP4 genes. Localization experiments showed that CaPGIP1, CaPGIP3, and CaPGIP4 are located in the cell wall or membrane. Transcript levels of CaPGIP1, CaPGIP3, and CaPGIP4 genes analyzed at untreated conditions show varied expression patterns analogous to other defense-related gene families. Interestingly, CaPGIP2 lacked a signal peptide, more than half of the LRRs, and other characteristics of a typical PGIP and subcellular localization indicated it is not located in the cell wall or membrane. The study's findings demonstrate CaPGIP1, CaPGIP3, and CaPGIP4's similarity to other legume PGIPs and suggest they might possess the potential to combat chickpea pathogens.

Draft genome data of Prunus avium cv 'Stella'.

Prunus avium cv. 'Stella' total cellular DNA was isolated from emerging leaf tissue and sequenced using Roche 454 GS FLX Titanium, and Illumina HiSeq 2000 High Throughput Sequencing (HTS) technologies. Sequence data were filtered and trimmed to retain nucleotides corresponding to Phred score 30, and assembled with CLC Genomics Workbench v.6.0.1. A total of 107,531 contigs were assembled with 185 scaffolds with a maximum length of 132,753 nucleotides and an N50 value of 4,601. The average depth of coverage was 135.87 nucleotides with a median depth of coverage equal to 31.50 nucleotides. The draft 'Stella' genome presented here covers 77.8% of the estimated 352.9Mb P. avium genome and is expected to facilitate genetics and genomics research focused on identifying genes and quantitative trait loci (QTL) underlying important agronomic and consumer traits.

Genome editing in fruit, ornamental, and industrial crops.

The advent of genome editing has opened new avenues for targeted trait enhancement in fruit, ornamental, industrial, and all specialty crops. In particular, CRISPR-based editing systems, derived from bacterial immune systems, have quickly become routinely used tools for research groups across the world seeking to edit plant genomes with a greater level of precision, higher efficiency, reduced off-target effects, and overall ease-of-use compared to ZFNs and TALENs. CRISPR systems have been applied successfully to a number of horticultural and industrial crops to enhance fruit ripening, increase stress tolerance, modify plant architecture, control the timing of flower development, and enhance the accumulation of desired metabolites, among other commercially-important traits. As editing technologies continue to advance, so too does the ability to generate improved crop varieties with non-transgenic modifications; in some crops, direct transgene-free edits have already been achieved, while in others, T-DNAs have successfully been segregated out through crossing. In addition to the potential to produce non-transgenic edited crops, and thereby circumvent regulatory impediments to the release of new, improved crop varieties, targeted gene editing can speed up trait improvement in crops with long juvenile phases, reducing inputs resulting in faster market introduction to the market. While many challenges remain regarding optimization of genome editing in ornamental, fruit, and industrial crops, the ongoing discovery of novel nucleases with niche specialties for engineering applications may form the basis for additional and potentially crop-specific editing strategies.

Genome editing reagent delivery in plants.

Genome editing holds the potential for rapid crop improvement to meet the challenge of feeding the planet in a changing climate. The delivery of gene editing reagents into the plant cells has been dominated by plasmid vectors delivered using agrobacterium or particle bombardment. This approach involves the production of genetically engineered plants, which need to undergo regulatory approvals. There are various reagent delivery approaches available that have enabled the delivery of DNA-free editing reagents. They invariably involve the use of ribonucleoproteins (RNPs), especially in the case of CRISPR/Cas9-mediated gene editing. The explant of choice for most of the non-DNA approaches utilizes protoplasts as the recipient explant. While the editing efficiency is high in protoplasts, the ability to regenerate individual plants from edited protoplasts remains a challenge. There are various innovative delivery approaches being utilized to perform in planta edits that can be incorporated in the germline cells or inherited via seed. With the modification and adoption of various novel approaches currently being used in animal systems, it seems likely that non-transgenic genome editing will become routine in higher plants.

Metabolic Engineering of Oleaginous Yeast Yarrowia lipolytica for Overproduction of Fatty Acids.

The oleaginous yeast Yarrowia lipolytica has attracted much attention due to its ability to utilize a wide range of substrates to accumulate high lipid content and its flexibility for genetic manipulation. In this study, intracellular lipid metabolism in Y. lipolytica was tailored to produce fatty acid, a renewable oleochemical and precursor for production of advanced biofuels. Two main strategies, including blocking activation and peroxisomal uptake of fatty acids and elimination of biosynthesis of lipids, were employed to reduce fatty acid consumption by the native pathways in Y. lipolytica. Both genetic modifications improved fatty acid production. However, disruption of the genes responsible for assembly of nonpolar lipid molecules including triacylglycerols (TAGs) and steryl esters resulted in the deleterious effects on the cell growth. The gene tesA encoding thioesterase from Escherichia coli was expressed in the strain with disrupted faa genes encoding fatty acyl-CoA synthetases and pxa1 encoding peroxisomal acyl-CoA transporter, and the titer of fatty acids resulted in 2.3 g/L in shake flask culture, representing 11-fold improvement compared with the parent strain. Expressing the native genes encoding acetyl-CoA carboxylase (ACC) and hexokinase also increased fatty acid production, although the improvement was not as significant as that with tesA expression. Saturated fatty acids including palmitic acid (C16:0) and stearic acid (C18:0) increased remarkably in the fatty acid composition of the recombinant bearing tesA compared with the parent strain. The recombinant expressing tesA gene resulted in high lipid content, indicating the great fatty acid producing potential of Y. lipolytica. The results highlight the achievement of fatty acid overproduction without adverse effect on growth of the strain. Results of this study provided insight into the relationship between fatty acid and lipid metabolism in Y. lipolytica, confirming the avenue to reprogram lipid metabolism of this host for overproduction of renewable fatty acids.

Glyoxylic acid overcomes 1-MCP-induced blockage of fruit ripening in Pyrus communis L. var. 'D'Anjou'.

1-methylcyclopropene (1-MCP) in an ethylene receptor antagonist that blocks ethylene perception and downstream ripening responses in climacteric fruit imparting a longer shelf life. However, in European pear, the application of 1-MCP irreversibly obstructs the onset of system 2 ethylene production resulting in perpetually unripe fruit with undesirable quality. Application of exogenous ethylene, carbon dioxide and treatment to high temperatures is not able to reverse the blockage in ripening. We recently reported that during cold conditioning, activation of alternative oxidase (AOX) occurs pre-climacterically. In this study, we report that activation of AOX via exposure of 1-MCP treated 'D'Anjou' pear fruit to glyoxylic acid triggers an accelerated ripening response. Time course physiological analysis revealed that ripening is evident from decreased fruit firmness and increased internal ethylene. Transcriptomic and functional enrichment analyses revealed genes and ontologies implicated in glyoxylic acid-mediated ripening, including AOX, TCA cycle, fatty acid metabolism, amino acid metabolism, organic acid metabolism, and ethylene-responsive pathways. These observations implicate the glyoxylate cycle as a biochemical hub linking multiple metabolic pathways to stimulate ripening through an alternate mechanism. The results provide information regarding how blockage caused by 1-MCP may be circumvented at the metabolic level, thus opening avenues for consistent ripening in pear and possibly other fruit.

CRISPR-associated nucleases: the Dawn of a new age of efficient crop improvement.

The world stands at a new threshold today. As a planet, we face various challenges, and the key one is how to continue to produce enough food, feed, fiber, and fuel to support the burgeoning population. In the past, plant breeding and the ability to genetically engineer crops contributed to increasing food production. However, both approaches rely on random mixing or integration of genes, and the process can be unpredictable and time-consuming. Given the challenge of limited availability of natural resources and changing environmental conditions, the need to rapidly and precisely improve crops has become urgent. The discovery of CRISPR-associated endonucleases offers a precise yet versatile platform for rapid crop improvement. This review summarizes a brief history of the discovery of CRISPR-associated nucleases and their application in genome editing of various plant species. Also provided is an overview of several new endonucleases reported recently, which can be utilized for editing of specific genes in plants through various forms of DNA sequence alteration. Genome editing, with its ever-expanding toolset, increased efficiency, and its potential integration with the emerging synthetic biology approaches hold promise for efficient crop improvement to meet the challenge of supporting the needs of future generations.

Exploring fatty alcohol-producing capability of Yarrowia lipolytica.

BACKGROUND: Fatty alcohols are important oleochemicals widely used in detergents, surfactants and personal care products. Bio-synthesized fatty alcohol provides a promising alternative to traditional fatty alcohol industry. Harnessing oleaginous microorganisms for fatty alcohol production may offer a new strategy to achieve a commercially viable yield that currently still seems to be a remote target. RESULTS: In this study, we introduced functional fatty acyl-CoA reductase (FAR), TaFAR1 to direct the conversion from fatty acyl-CoA to fatty alcohol in Yarrowia lipolytica (Y. lipolytica), an oleaginous non-conventional yeast showing great lipid-producing capability. Tri-module optimizations including eliminating fatty alcohol degradation pathway, enhancing TaFAR1 expression, and increasing fatty acyl-CoA supply were furtherly conducted, resulting in 63-fold increase in intracellular fatty alcohol-producing capability compared to the starting strain. Thus, this work demonstrated successful construction of first generation of Y. lipolytica fatty alcohol-producing cell factory. Through the study of effect of environmental nutrition on fatty alcohol production, up to 636.89 mg/L intracellular hexadecanol (high fatty alcohol-retaining capability) and 53.32 mg/L extracellular hexadecanol were produced by this cell factory through batch fermentation, which was comparable to the highest production of Saccharomyces cerevisiae under the similar condition. CONCLUSION: This work preliminarily explored fatty alcohol-producing capability through mobilization of FAR and fatty acid metabolism, maximizing the intracellular fatty alcohol-producing capability, suggesting that Y. lipolytica cell factory potentially offers a promising platform for fatty alcohol production.