Development of a Sensitive, Easy and High-Throughput Compliant Protocol for Maize and Soybean DNA Extraction and Quantitation Using a Plant-Specific Universal Taqman Minor Groove Binder Probe.

We report the optimization of a high-throughput, compliant DNA extraction method that uses standard format 96-well plates and a commercial automated DNA purification system (ABI PRISM® 6100 Nucleic Acid PrepStation). The procedure was set up for maize and soybean, the most common GMO crops and the main ingredients of several foodstuffs, and compared with an EU-validated CTAB-based method. Optimization of the DNA extraction was achieved by applying self-prepared buffers (for DNA extraction, binding, and washing) on the PrepStation loaded with proprietary glass-fiber-coated purification plates. Quantification of extracted DNA was performed by real-time PCR using previously reported endogenous soybean lectin and maize starch synthase genes and a novel plant-specific universal TaqMan MGB probe that targets the 18S rRNA multiple copy gene. Using serial dilutions of both maize and soybean genomic DNAs, we show low PCR sensitivity and efficiency for the official TransPrep DNA extraction protocol compared to the CTAB-based one. On the other hand, using serial dilutions of a standard reference plasmid containing a 137 bp sequence cloned from the 18S rRNA plant-specific ribosomal gene, we demonstrate the high PCR sensitivity and efficiency of the optimized DNA extraction protocol setup with self-prepared buffers. The limits of detection and quantification of the 18S rDNA reiteration were consistent with the calculated values, supporting the suitability of the DNA extraction procedure for high-throughput analyses of large populations and small amounts of tissue.

Exploring the metabolic and physiological roles of HQT in S. lycopersicum by gene editing.

The most abundant phenolic compound in Solanaceous plants is chlorogenic acid (CGA), which possesses protective properties such as antimicrobial and antioxidant activities. These properties are particularly relevant when plants are under adverse conditions, such as pathogen attack, excess light, or extreme temperatures that cause oxidative stress. Additionally, CGA has been shown to absorb UV-B light. In tomato and potato, CGA is mainly produced through the HQT pathway mediated by the enzyme hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase. However, the absence of natural or induced mutants of this gene has made it unclear whether other pathways contribute to CGA production and accumulation. To address this question, we used CRISPR technology to generate multiple knock-out mutant lines in the tomato HQT gene. The resulting slhqt plants did not accumulate CGA or other caffeoylquinic acids (CQAs) in various parts of the plant, indicating that CQA biosynthesis depends almost entirely on the HQT pathway in tomato and, likely, other Solanaceous crops. We also found that the lack of CGA in slhqt plants led to higher levels of hydroxycinnamoyl-glucose and flavonoids compared to wild-type plants. Gene expression analysis revealed that this metabolic reorganization was partly due to flux redirection, but also involved modulation of important transcription factor genes that regulate secondary metabolism and sense environmental conditions. Finally, we investigated the physiological role of CGA in tomato and found that it accumulates in the upper epidermis where it acts as a protector against UV-B irradiation.

Targeted mutagenesis of StISAC stabilizes the production of anthocyanins in potato cell culture.

To increase the production of decorated anthocyanins in potato cell cultures, we knocked out a novel potato gene, named Inducer Silencing of Anthocyanins in Cell culture (StISAC), using CRISPR-Cas9 editing. Our results provided evidence that mutant cell lines doubled the accumulation level of anthocyanins biosynthesized. Moreover, the production of these important pigments was stabilized over time. Our study overcame important challenges in the efficient biotechnological production of these valuable pigments and reported the function of a novel anthocyanin biosynthesis repressor gene.

Biofortified tomatoes provide a new route to vitamin D sufficiency.

Poor vitamin D status is a global health problem; insufficiency underpins higher risk of cancer, neurocognitive decline and all-cause mortality. Most foods contain little vitamin D and plants are very poor sources. We have engineered the accumulation of provitamin D3 in tomato by genome editing, modifying a duplicated section of phytosterol biosynthesis in Solanaceous plants, to provide a biofortified food with the added possibility of supplement production from waste material.

Conservation of AtTZF1, AtTZF2, and AtTZF3 homolog gene regulation by salt stress in evolutionarily distant plant species.

Arginine-rich tandem zinc-finger proteins (RR-TZF) participate in a wide range of plant developmental processes and adaptive responses to abiotic stress, such as cold, salt, and drought. This study investigates the conservation of the genes AtTZF1-5 at the level of their sequences and expression across plant species. The genomic sequences of the two RR-TZF genes TdTZF1-A and TdTZF1-B were isolated in durum wheat and assigned to chromosomes 3A and 3B, respectively. Sequence comparisons revealed that they encode proteins that are highly homologous to AtTZF1, AtTZF2, and AtTZF3. The expression profiles of these RR-TZF durum wheat and Arabidopsis proteins support a common function in the regulation of seed germination and responses to abiotic stress. In particular, analysis of plants with attenuated and overexpressed AtTZF3 indicate that AtTZF3 is a negative regulator of seed germination under conditions of salt stress. Finally, comparative sequence analyses establish that the RR-TZF genes are encoded by lower plants, including the bryophyte Physcomitrella patens and the alga Chlamydomonas reinhardtii. The regulation of the Physcomitrella AtTZF1-2-3-like genes by salt stress strongly suggests that a subgroup of the RR-TZF proteins has a function that has been conserved throughout evolution.

Detection and quantification of genetically modified organisms using very short, locked nucleic acid TaqMan probes.

Many countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (PCR) based upon the TaqMan probe chemistry has become the method mostly used to support these regulations; moreover, event-specific PCR is the preferred method in GMO detection because of its high specificity based on the flanking sequence of the exogenous integrant. The aim of this study was to evaluate the use of very short (eight-nucleotide long), locked nucleic acid (LNA) TaqMan probes in 5'-nuclease PCR assays for the detection and quantification of GMOs. Classic TaqMan and LNA TaqMan probes were compared for the analysis of the maize MON810 transgene. The performance of the two types of probes was tested on the maize endogenous reference gene hmga, the CaMV 35S promoter, and the hsp70/cryIA(b) construct as well as for the event-specific 5'-integration junction of MON810, using plasmids as standard reference molecules. The results of our study demonstrate that the LNA 5'-nuclease PCR assays represent a valid and reliable analytical system for the detection and quantification of transgenes. Application of very short LNA TaqMan probes to GMO quantification can simplify the design of 5'-nuclease assays.