Drought Resistance by Engineering Plant Tissue-Specific Responses

Drought is the primary cause of agricultural loss globally, and represents a major threat to food security. Currently, plant biotechnology stands as one of the most promising fields when it comes to developing crops that are able to produce high yields in water-limited conditions. From studies of Arabidopsis thaliana whole plants, the main response mechanisms to drought stress have been uncovered, and multiple drought resistance genes have already been engineered into crops. So far, most plants with enhanced drought resistance have displayed reduced crop yield, meaning that there is still a need to search for novel approaches that can uncouple drought resistance from plant growth. Our laboratory has recently shown that the receptors of brassinosteroid (BR) hormones use tissue-specific pathways to mediate different developmental responses during root growth. In Arabidopsis, we found that increasing BR receptors in the vascular plant tissues confers resistance to drought without penalizing growth, opening up an exceptional opportunity to investigate the mechanisms that confer drought resistance with cellular specificity in plants. In this review, we provide an overview of the most promising phenotypical drought traits that could be improved biotechnologically to obtain drought-tolerant cereals. In addition, we discuss how current genome editing technologies could help to identify and manipulate novel genes that might grant resistance to drought stress. In the upcoming years, we expect that sustainable solutions for enhancing crop production in water-limited environments will be identified through joint efforts

CRISPR-Cas9 Multiplex Editing of the α-Amylase/Trypsin Inhibitor Genes to Reduce Allergen Proteins in Durum Wheat

Wheat and its derived foods are widespread, representing one of the main food sources globally. During the last decades, the incidence of disorders related to wheat has become a global issue for the human population, probably linked to the spread of wheat-derived foods. It has been ascertained that structural and metabolic proteins, like \u3b1-amylase/trypsin inhibitors (ATI), are involved in the onset of wheat allergies (bakers' asthma) and probably Non-Coeliac Wheat Sensitivity (NCWS). The ATI are a group of exogenous protease inhibitors, which are encoded by a multigene family dispersed over several chromosomes in durum and bread wheat. WTAI-CM3 and WTAI-CM16 subunits are considered among the main proteins involved in the onset of bakers' asthma and probably NCWS. A CRISPR-Cas9 multiplexing strategy was used to edit the ATI subunits WTAI-CM3 and WTAI-CM16 in the grain of the Italian durum wheat cultivar Svevo with the aim to produce wheat lines with reduced amount of potential allergens involved in adverse reactions. Using a marker gene-free approach, whereby plants are regenerated without selection agents, homozygous mutant plants without the presence of CRISPR vectors were obtained directly from T0 generation. This study demonstrates the capability of CRISPR technology to knock out immunogenic proteins in a reduced time compared to conventional breeding programmes. The editing of the two target genes was confirmed either at molecular (sequencing and gene expression study) or biochemical (immunologic test) level. Noteworthy, as a pleiotropic effect, is the activation of the ATI 0.28 pseudogene in the edited lines

Efficacy and safety of honeybee and wasp tyrosine-adsorbed venom immunotherapy

It is acknowledged that any claim of efficacy of allergen immunotherapy must be done for each specific product, and this remains true also for venom immunotherapy (VIT). Thus, we evaluated the efficacy and safety of a specific tyrosine-adsorbed VIT for vespula spp. and honeybee in real-life

Wheat with greatly reduced accumulation of free asparagine in the grain, produced by CRISPR/Cas9 editing of asparagine synthetase gene TaASN2

Free asparagine is the precursor for acrylamide, which forms during the baking, toasting and high-temperature processing of foods made from wheat. In this study, CRISPR/Cas9 was used to knock out the asparagine synthetase gene, TaASN2, of wheat (Triticum aestivum) cv. Cadenza. A 4-gRNA polycistronic gene was introduced into wheat embryos by particle bombardment and plants were regenerated. T1 plants derived from 11 of 14 T0 plants were shown to carry edits. Most edits were deletions (up to 173 base pairs), but there were also some single base pair insertions and substitutions. Editing continued beyond the T1 generation. Free asparagine concentrations in the grain of plants carrying edits in all six TaASN2 alleles (both alleles in each genome) were substantially reduced compared with wildtype, with one plant showing a more than 90 % reduction in the T2 seeds. A plant containing edits only in the A genome alleles showed a smaller reduction in free asparagine concentration in the grain, but the concentration was still lower than in wildtype. Free asparagine concentration in the edited plants was also reduced as a proportion of the free amino acid pool. Free asparagine concentration in the T3 seeds remained substantially lower in the edited lines than wildtype, although it was higher than in the T2 seeds, possibly due to stress. In contrast, the concentrations of free glutamine, glutamate and aspartate were all higher in the edited lines than wildtype. Low asparagine seeds showed poor germination but this could be overcome by exogenous application of asparagine

Antagonistic Transcription Factor Complexes Modulate the Floral Transition in Rice

Plants measure day or night lengths to coordinate specific developmental changes with a favorable season. In rice (Oryza sativa), the reproductive phase is initiated by exposure to short days when expression of HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1) is induced in leaves. The cognate proteins are components of the florigenic signal and move systemically through the phloem to reach the shoot apical meristem (SAM). In the SAM, they form a transcriptional activation complex with the bZIP transcription factor OsFD1 to start panicle development. Here, we show that Hd3a and RFT1 can form transcriptional activation or repression complexes also in leaves and feed back to regulate their own transcription. Activation complexes depend on OsFD1 to promote flowering. However, additional bZIPs, including Hd3a BINDING REPRESSOR FACTOR1 (HBF1) and HBF2, form repressor complexes that reduce Hd3a and RFT1 expression to delay flowering. We propose that Hd3a and RFT1 are also active locally in leaves to fine-tune photoperiodic flowering response

A new radiative microfurnace for X-ray single-crystal diffractometry

Microfurnaces available today for X-ray single-crystal investigation (radiative, gas-flame, gas-flow) are generally temperature-monitored by a thermocouple placed in a different site with respect to the crystal. The new F1 microfurnace aims at the following goals: fast crystal mounting; access to a large portion of reciprocal lattice; low absorption of direct beam; efficient in situ temperature measurement; fast power supply regulation; high temperature stability; controlled atmosphere. In the F1, the crystal is directly glued to a thermocouple with refractory cement. The heating body, fixed on the chi and radially shiftable along the phi axis circle, hosts the Pt winding and connections of both gas flow and power supply, and is enclosed in a thin pyrolitic boron nitride (PBN) shield. Temperature stability is within +/- 1 degreesC. Calibration of the F1 was carried out by collecting X-ray data from a single-crystal synthetic periclase at temperatures: of 28, 150, 300, 450, 600, 700, 900 and 1000 degreesC, and yielded the equation: a = 0.0000625 (7) [T(K) - 273] + 4.2083 (4), based on the ratio between cell edge and temperature. Structural refinements gave structural parameters and thermal expansion values at the investigated temperature. The mean coefficient of linear expansion up to 1000 degreesC was 14.3 x 10(-6) degreesC

Abscisic Acid and Flowering Regulation : Many Targets, Different Places

Plants can react to drought stress by anticipating flowering, an adaptive strategy for plant survival in dry climates known as drought escape (DE). In Arabidopsis, the study of DE brought to surface the involvement of abscisic acid (ABA) in controlling the floral transition. A central question concerns how and in what spatial context can ABA signals affect the floral network. In the leaf, ABA signaling affects flowering genes responsible for the production of the main florigen FLOWERING LOCUS T (FT). At the shoot apex, FD and FD-like transcription factors interact with FT and FT-like proteins to regulate ABA responses. This knowledge will help separate general and specific roles of ABA signaling with potential benefits to both biology and agriculture

Analisi e miglioramento delle prestazioni della Lean Experience Factory

Il lavoro intende presentare le analisi effettuate mediante modello simulativo della "Lean Experience Factory" sia nella configurazione iniziale che in quella lean, evidenziando l'ottenimento degli stessi indici di prestazione rispetto alle misurazioni svolte nell'ambiente reale. Lo sviluppo di un modello di simulazione "affidabile" consentir\ue0 di testare delle ulteriori configurazioni, che permetteranno di migliorare ulteriormente le prestazioni della fabbrica modello e di testare alcune condizioni limite del modello. Esse potranno essere applicate nelle realt\ue0 industriali

Unraveling the regulatory pathway of lysine-specific histone demethylases in plants

""In Arabidopsis thaliana four lysine-specific histone demethylases were recently identified (AtLSD1, AtLSD2, AtLSD3 and AtLSD4). These proteins participate in epigenetic regulation of gene expression in association with multi-protein complexes and are involved in important developmental processes, such as flower transition and root elongation. . To identify AtLSDs molecular partners, Arabidopsis plants were transformed with a 35S::AtLSD1-FLAG-HA construct and recombinant AtLSD1 together with associated proteins were immunoprecipitated. Mass spectrometry-based analysis of these proteins leads to interesting insights. Phenotypical analysis of loss-of-function atlsd mutants and 35S::AtLSDs transgenic plants under both physiological and stress conditions is in progress. These mutant plants are also analyzed for the expression of genes associated to plant developmental programs and defense resposes to determine the AtLSD specific targets. To obtain information on the tissue- and organ-specific expression pattern of the AtLSDs, AtLSD::GFP-GUS were obtained. Using this multi-disciplinary approach it has been possible to highlight important differences among the various AtLSDs.. "