Is CRISPR/Cas9-based multi-trait enhancement of wheat forthcoming?

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies have been implemented in recent years in the genome editing of eukaryotes, including plants. The original system of knocking out a single gene by causing a double-strand break (DSB), followed by non-homologous end joining (NHEJ) or Homology-directed repair (HDR) has undergone many adaptations. These adaptations include employing CRISPR/Cas9 to upregulate gene expression or to cause specific small changes to the DNA sequence of the gene-of-interest. In plants, multiplexing, i.e., inducing multiple changes by CRISPR/Cas9, is extremely relevant due to the redundancy of many plant genes, and the time- and labor-consuming generation of stable transgenic plant lines via crossing. Here we discuss relevant examples of various traits, such as yield, biofortification, gluten content, abiotic stress tolerance, and biotic stress resistance, which have been successfully manipulated using CRISPR/Cas9 in plants. While existing studies have primarily focused on proving the impact of CRISPR/Cas9 on a single trait, there is a growing interest among researchers in creating a multi-stress tolerant wheat cultivar 'super wheat', to commercially and sustainably enhance wheat yields under climate change. Due to the complexity of the technical difficulties in generating multi-target CRISPR/Cas9 lines and of the interactions between stress responses, we propose enhancing already commercial local landraces with higher yield traits along with stress tolerances specific to the respective localities, instead of generating a general 'super wheat'. We hope this will serve as the sustainable solution to commercially enhancing crop yields under both stable and challenging environmental conditions.

The Alteration of Tomato Chloroplast Vesiculation Positively Affects Whole-Plant Source-Sink Relations and Fruit Metabolism under Stress Conditions.

Changes in climate conditions can negatively affect the productivity of crop plants. They can induce chloroplast degradation (senescence), which leads to decreased source capacity, as well as decreased whole-plant carbon/nitrogen assimilation and allocation. The importance, contribution and mechanisms of action regulating source-tissue capacity under stress conditions in tomato (Solanum lycopersicum) are not well understood. We hypothesized that delaying chloroplast degradation by altering the activity of the tomato chloroplast vesiculation (CV) under stress would lead to more efficient use of carbon and nitrogen and to higher yields. Tomato CV is upregulated under stress conditions. Specific induction of CV in leaves at the fruit development stage resulted in stress-induced senescence and negatively affected fruit yield, without any positive effects on fruit quality. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/CAS9) knockout CV plants, generated using a near-isogenic tomato line with enhanced sink capacity, exhibited stress tolerance at both the vegetative and the reproductive stages, leading to enhanced fruit quantity, quality and harvest index. Detailed metabolic and transcriptomic network analysis of sink tissue revealed that the l-glutamine and l-arginine biosynthesis pathways are associated with stress-response conditions and also identified putative novel genes involved in tomato fruit quality under stress. Our results are the first to demonstrate the feasibility of delayed stress-induced senescence as a stress-tolerance trait in a fleshy fruit crop, to highlight the involvement of the CV pathway in the regulation of source strength under stress and to identify genes and metabolic pathways involved in increased tomato sink capacity under stress conditions.

The effect of circular soil biosolarization treatment on the physiology, metabolomics, and microbiome of tomato plants under certain abiotic stresses.

Soil biosolarization (SBS) is an alternative technique for soil pest control to standard techniques such as soil fumigation and soil solarization (SS). By using both solar heating and fermentation of organic amendments, faster and more effective control of soilborne pathogens can be achieved. A circular economy may be created by using the residues of a given crop as organic amendments to biosolarize fields that produce that crop, which is termed circular soil biosolarization (CSBS). In this study, CSBS was employed by biosolarizing soil with amended tomato pomace (TP) residues and examining its impact on tomato cropping under conditions of abiotic stresses, specifically high salinity and nitrogen deficiency. The results showed that in the absence of abiotic stress, CSBS can benefit plant physiological performance, growth and yield relative to SS. Moreover, CSBS significantly mitigated the impacts of abiotic stress conditions. The results also showed that CSBS impacted the soil microbiome and plant metabolome. Mycoplana and Kaistobacter genera were found to be positively correlated with benefits to tomato plants health under abiotic stress conditions. Conversely, the relative abundance of the orders RB41, MND1, and the family Ellin6075 and were negatively correlated with tomato plants health. Moreover, several metabolites were significantly affected in plants grown in SS- and CSBS-treated soils under abiotic stress conditions. The metabolite xylonic acid isomer was found to be significantly negatively correlated with tomato plants health performance across all treatments. These findings improve understanding of the interactions between CSBS, soil ecology, and crop physiology under abiotic stress conditions.

Resolving diurnal dynamics of the chloroplastic glutathione redox state in Arabidopsis reveals its photosynthetically derived oxidation.

Plants are subjected to fluctuations in light intensity, and this might cause unbalanced photosynthetic electron fluxes and overproduction of reactive oxygen species (ROS). Electrons needed for ROS detoxification are drawn, at least partially, from the cellular glutathione (GSH) pool via the ascorbate-glutathione cycle. Here, we explore the dynamics of the chloroplastic glutathione redox potential (chl-EGSH) using high-temporal-resolution monitoring of Arabidopsis (Arabidopsis thaliana) lines expressing the reduction-oxidation sensitive green fluorescent protein 2 (roGFP2) in chloroplasts. This was carried out over several days under dynamic environmental conditions and in correlation with PSII operating efficiency. Peaks in chl-EGSH oxidation during dark-to-light and light-to-dark transitions were observed. Increasing light intensities triggered a binary oxidation response, with a threshold around the light saturating point, suggesting two regulated oxidative states of the chl-EGSH. These patterns were not affected in npq1 plants, which are impaired in non-photochemical quenching. Oscillations between the two oxidation states were observed under fluctuating light in WT and npq1 plants, but not in pgr5 plants, suggesting a role for PSI photoinhibition in regulating the chl-EGSH dynamics. Remarkably, pgr5 plants showed an increase in chl-EGSH oxidation during the nights following light stresses, linking daytime photoinhibition and nighttime GSH metabolism. This work provides a systematic view of the dynamics of the in vivo chloroplastic glutathione redox state during varying light conditions.