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.

Null mutants of a tomato Rho of plants exhibit enhanced water use efficiency without a penalty to yield.

Improving water use efficiency in crops is a significant challenge as it involves balancing water transpiration and CO2 uptake through stomatal pores. This study investigates the role of SlROP9, a tomato Rho of Plants protein, in guard cells and its impact on plant transpiration. The results reveal that SlROP9 null mutants exhibit reduced stomatal conductance while photosynthetic CO2 assimilation remains largely unaffected. Notably, there is a notable decrease in whole-plant transpiration in the rop9 mutants compared to the wild type, especially during noon hours when the water pressure deficit is high. The elevated stomatal closure observed in rop9 mutants is linked to an increase in reactive oxygen species formation. This is very likely dependent on the respiratory burst oxidase homolog (RBOH) NADPH oxidase and is not influenced by abscisic acid (ABA). Consistently, activated ROP9 can interact with RBOHB in both yeast and plants. In diverse tomato accessions, drought stress represses ROP9 expression, and in Arabidopsis stomatal guard cells, ABA suppresses ROP signaling. Therefore, the phenotype of the rop9 mutants may arise from a disruption in ROP9-regulated RBOH activity. Remarkably, large-scale field experiments demonstrate that the rop9 mutants display improved water use efficiency without compromising fruit yield. These findings provide insights into the role of ROPs in guard cells and their potential as targets for enhancing water use efficiency in crops.

Dynamic metabolite QTL analyses provide novel biochemical insights into kernel development and nutritional quality improvement in common wheat.

Despite recent advances in crop metabolomics, the genetic control and molecular basis of the wheat kernel metabolome at different developmental stages remain largely unknown. Here, we performed widely targeted metabolite profiling of kernels from three developmental stages (grain-filling kernels [FKs], mature kernels [MKs], and germinating kernels [GKs]) using a population of 159 recombinant inbred lines. We detected 625 annotated metabolites and mapped 3173, 3143, and 2644 metabolite quantitative trait loci (mQTLs) in FKs, MKs, and GKs, respectively. Only 52 mQTLs were mapped at all three stages, indicating the high stage specificity of the wheat kernel metabolome. Four candidate genes were functionally validated by in vitro enzymatic reactions and/or transgenic approaches in wheat, three of which mediated the tricin metabolic pathway. Metabolite flux efficiencies within the tricin pathway were evaluated, and superior candidate haplotypes were identified, comprehensively delineating the tricin metabolism pathway in wheat. Finally, additional wheat metabolic pathways were re-constructed by updating them to incorporate the 177 candidate genes identified in this study. Our work provides new information on variations in the wheat kernel metabolome and important molecular resources for improvement of wheat nutritional quality.

The reference genome and abiotic stress responses of the model perennial grass Brachypodium sylvaticum.

Perennial grasses are important forage crops and emerging biomass crops and have the potential to be more sustainable grain crops. However, most perennial grass crops are difficult experimental subjects due to their large size, difficult genetics, and/or their recalcitrance to transformation. Thus, a tractable model perennial grass could be used to rapidly make discoveries that can be translated to perennial grass crops. Brachypodium sylvaticum has the potential to serve as such a model because of its small size, rapid generation time, simple genetics, and transformability. Here, we provide a high-quality genome assembly and annotation for B. sylvaticum, an essential resource for a modern model system. In addition, we conducted transcriptomic studies under 4 abiotic stresses (water, heat, salt, and freezing). Our results indicate that crowns are more responsive to freezing than leaves which may help them overwinter. We observed extensive transcriptional responses with varying temporal dynamics to all abiotic stresses, including classic heat-responsive genes. These results can be used to form testable hypotheses about how perennial grasses respond to these stresses. Taken together, these results will allow B. sylvaticum to serve as a truly tractable perennial model system.

Plasma membrane aquaporins regulate root hydraulic conductivity in the model plant Setaria viridis.

The high rate of productivity observed in panicoid crops is in part due to their extensive root system. Recently, green foxtail (Setaria viridis) has emerged as a genetic model system for panicoid grasses. Natural accessions of S. viridis originating from different parts of the world, with differential leaf physiological behavior, have been identified. This work focused on understanding the physiological and molecular mechanisms controlling root hydraulic conductivity and root-to-shoot gas exchange signaling in S. viridis. We identified 2 accessions, SHA and ZHA, with contrasting behavior at the leaf, root, and whole-plant levels. Our results indicated a role for root aquaporin (AQP) plasma membrane (PM) intrinsic proteins in the differential behavior of SHA and ZHA. Moreover, a different root hydraulic response to low levels of abscisic acid between SHA and ZHA was observed, which was associated with root AQPs. Using cell imaging, biochemical, and reverse genetic approaches, we identified PM intrinsic protein 1;6 (PIP1;6) as a possible PIP1 candidate that regulates radial root hydraulics and root-to-shoot signaling of gas exchange in S. viridis. In heterologous systems, PIP1;6 localized in the endoplasmic reticulum, and upon interaction with PIP2s, relocalization to the PM was observed. PIP1;6 was predominantly expressed at the root endodermis. Generation of knockout PIP1;6 plants (KO-PIP1;6) in S. viridis showed altered root hydraulic conductivity, altered gas exchange, and alteration of root transcriptional patterns. Our results indicate that PIPs are essential in regulating whole-plant water homeostasis in S. viridis. We conclude that root hydraulic conductivity and gas exchange are positively associated and are regulated by AQPs.

Limitations and advantages of using metabolite-based genome-wide association studies: Focus on fruit quality traits.

In the last decades, linkage mapping has help in the location of metabolite quantitative trait loci (QTL) in many species; however, this approach shows some limitations. Recently, thanks to the most recent advanced in high-throughput genotyping technologies like next-generation sequencing, metabolite genome-wide association study (mGWAS) has been proposed a powerful tool to identify the genetic variants in polygenic agrinomic traits. Fruit flavor is a complex interaction of aroma volatiles and taste being sugar and acid ratio key parameter for flavor acceptance. Here, we review recent progress of mGWAS in pinpoint gene polymorphisms related to flavor-related metabolites in fruits. Despite clear successes in discovering novel genes or regions associated with metabolite accumulation affecting sensory attributes in fruits, GWAS incurs in several limitations summarized in this review. In addition, in our own work, we performed mGWAS on 194 Citrus grandis accessions to investigate the genetic control of individual primary and lipid metabolites in ripe fruit. We have identified a total of 667 associations for 14 primary metabolites including amino acids, sugars, and organic acids, and 768 associations corresponding to 47 lipids. Furthermore, candidate genes related to important metabolites related to fruit quality such as sugars, organic acids and lipids were discovered.

Sounds emitted by plants under stress are airborne and informative.

Stressed plants show altered phenotypes, including changes in color, smell, and shape. Yet, airborne sounds emitted by stressed plants have not been investigated before. Here we show that stressed plants emit airborne sounds that can be recorded from a distance and classified. We recorded ultrasonic sounds emitted by tomato and tobacco plants inside an acoustic chamber, and in a greenhouse, while monitoring the plant's physiological parameters. We developed machine learning models that succeeded in identifying the condition of the plants, including dehydration level and injury, based solely on the emitted sounds. These informative sounds may also be detectable by other organisms. This work opens avenues for understanding plants and their interactions with the environment and may have significant impact on agriculture.

Guard cell activity of PIF4 and HY5 control transpiration.

Whole-plant transpiration, controlled by plant hydraulics and stomatal movement, is regulated by endogenous and environmental signals, with the light playing a dominant role. Stomatal pore size continuously adjusts to changes in light intensity and quality to ensure optimal CO2 intake for photosynthesis on the one hand, together with minimal water loss on the other. The link between light and transpiration is well established, but the genetic knowledge of how guard cells perceive those signals to affect stomatal conductance is still somewhat limited. In the current study, we evaluated the role of two central light-responsive transcription factors; a bZIP-family transcription factor ELONGATED HYPOCOTYL5 (HY5) and the basic helix-loop-helix (BHLH) transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4), in the regulation of steady-state transpiration. We show that overexpression of PIF4 exclusively in guard cells (GCPIF4) decreases transpiration, and can restrain the high transpiration of the pif4 mutant. Expression of HY5 specifically in guard cells (GCHY5) had the opposite effect of enhancing transpiration rates of WT- Arabidopsis and tobacco plants and of the hy5 mutant in Arabidopsis. In addition, we show that GCHY5 can reverse the low transpiration caused by guard cell overexpression of the sugar sensor HEXOKINASE1 (HXK1, GCHXK), an established low transpiring genotype. Finally, we suggest that the GCHY5 reversion of low transpiration by GCHXK requires the auto-activation of the endogenous HY5 in other tissues. These findings support the existence of an ongoing diurnal regulation of transpiration by the light-responsive transcription factors HY5 and PIF4 in the stomata, which ultimately determine the whole-plant water use efficiency.

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.

ABA homeostasis and long-distance translocation are redundantly regulated by ABCG ABA importers.

The effects of abscisic acid (ABA) on plant growth, development, and response to the environment depend on local ABA concentrations. Here, we show that in Arabidopsis, ABA homeostasis is regulated by two previously unknown ABA transporters. Adenosine triphosphate­binding cassette subfamily G member 17 (ABCG17) and ABCG18 are localized to the plasma membranes of leaf mesophyll and cortex cells to redundantly promote ABA import, leading to conjugated inactive ABA sinks, thus restricting stomatal closure. ABCG17 and ABCG18 double knockdown revealed that the transporters encoded by these genes not only limit stomatal aperture size, conductance, and transpiration while increasing water use efficiency but also control ABA translocation from the shoot to the root to regulate lateral root emergence. Under abiotic stress conditions, ABCG17 and ABCG18 are transcriptionally repressed, promoting active ABA movement and response. The transport mechanism mediated by ABCG17 and ABCG18 allows plants to maintain ABA homeostasis under normal growth conditions.

Genomic basis underlying the metabolome-mediated drought adaptation of maize.

BACKGROUND: Drought is a major environmental disaster that causes crop yield loss worldwide. Metabolites are involved in various environmental stress responses of plants. However, the genetic control of metabolomes underlying crop environmental stress adaptation remains elusive. RESULTS: Here, we perform non-targeted metabolic profiling of leaves for 385 maize natural inbred lines grown under well-watered as well as drought-stressed conditions. A total of 3890 metabolites are identified and 1035 of these are differentially produced between well-watered and drought-stressed conditions, representing effective indicators of maize drought response and tolerance. Genetic dissections reveal the associations between these metabolites and thousands of single-nucleotide polymorphisms (SNPs), which represented 3415 metabolite quantitative trait loci (mQTLs) and 2589 candidate genes. 78.6% of mQTLs (2684/3415) are novel drought-responsive QTLs. The regulatory variants that control the expression of the candidate genes are revealed by expression QTL (eQTL) analysis of the transcriptomes of leaves from 197 maize natural inbred lines. Integrated metabolic and transcriptomic assays identify dozens of environment-specific hub genes and their gene-metabolite regulatory networks. Comprehensive genetic and molecular studies reveal the roles and mechanisms of two hub genes, Bx12 and ZmGLK44, in regulating maize metabolite biosynthesis and drought tolerance. CONCLUSION: Our studies reveal the first population-level metabolomes in crop drought response and uncover the natural variations and genetic control of these metabolomes underlying crop drought adaptation, demonstrating that multi-omics is a powerful strategy to dissect the genetic mechanisms of crop complex traits.

Guard cells control hypocotyl elongation through HXK1, HY5, and PIF4.

The hypocotyls of germinating seedlings elongate in a search for light to enable autotrophic sugar production. Upon exposure to light, photoreceptors that are activated by blue and red light halt elongation by preventing the degradation of the hypocotyl-elongation inhibitor HY5 and by inhibiting the activity of the elongation-promoting transcription factors PIFs. The question of how sugar affects hypocotyl elongation and which cell types stimulate and stop that elongation remains unresolved. We found that overexpression of a sugar sensor, Arabidopsis hexokinase 1 (HXK1), in guard cells promotes hypocotyl elongation under white and blue light through PIF4. Furthermore, expression of PIF4 in guard cells is sufficient to promote hypocotyl elongation in the light, while expression of HY5 in guard cells is sufficient to inhibit the elongation of the hy5 mutant and the elongation stimulated by HXK1. HY5 exits the guard cells and inhibits hypocotyl elongation, but is degraded in the dark. We also show that the inhibition of hypocotyl elongation by guard cells' HY5 involves auto-activation of HY5 expression in other tissues. It appears that guard cells are capable of coordinating hypocotyl elongation and that sugar and HXK1 have the opposite effect of light on hypocotyl elongation, converging at PIF4.

Arabidopsis leaf hydraulic conductance is regulated by xylem sap pH, controlled, in turn, by a P-type H+ -ATPase of vascular bundle sheath cells.

The leaf vascular bundle sheath cells (BSCs) that tightly envelop the leaf veins, are a selective and dynamic barrier to xylem sap water and solutes radially entering the mesophyll cells. Under normal conditions, xylem sap pH below 6 is presumably important for driving and regulating the transmembranal solute transport. Having discovered recently a differentially high expression of a BSC proton pump, AHA2, we now test the hypothesis that it regulates the xylem sap pH and leaf radial water fluxes. We monitored the xylem sap pH in the veins of detached leaves of wild-type Arabidopsis, AHA mutants and aha2 mutants complemented with AHA2 gene solely in BSCs. We tested an AHA inhibitor (vanadate) and stimulator (fusicoccin), and different pH buffers. We monitored their impact on the xylem sap pH and the leaf hydraulic conductance (Kleaf ), and the effect of pH on the water osmotic permeability (Pf ) of isolated BSCs protoplasts. We found that AHA2 is necessary for xylem sap acidification, and in turn, for elevating Kleaf . Conversely, AHA2 knockdown, which alkalinized the xylem sap, or, buffering its pH to 7.5, reduced Kleaf , and elevating external pH to 7.5 decreased the BSCs Pf . All these showed a causative link between AHA2 activity in BSCs and leaf radial hydraulic water conductance.

A Cytoplasmic Receptor-like Kinase Contributes to Salinity Tolerance.

Receptor-like cytoplasmic kinases (RLCKs) are receptor kinases that lack extracellular ligand-binding domains and have emerged as a major class of signaling proteins that regulate plant cellular activities in response to biotic/abiotic stresses and endogenous extracellular signaling molecules. We have identified a rice RLCK (OsRLCK311) that was significantly higher in transgenic pSARK-IPT rice (Oryza sativa) that exhibited enhanced growth under saline conditions. Overexpression of OsRLCK311 full-length protein (RLCK311FL) and the C-terminus of OsRLCK311 (ΔN) in Arabidopsis confirmed its role in salinity tolerance, both in seedlings and mature plants. Protein interaction assays indicated that OsRLCK311 and ΔN interacted in-vivo with the plasma membrane AQP AtPIP2;1. The RLCK311-PIP2;1 binding led to alterations in the stomata response to ABA, which was characterized by more open stomata of transgenic plants. Moreover, OsRLCK311-ΔN effect in mediating enhanced plant growth under saline conditions was also observed in the perennial grass Brachypodium sylvaticum, confirming its role in both dicots and monocots species. Lastly, OsRLCK311 interacted with the rice OsPIP2;1. We suggest that the rice OsRLCK311 play a role in regulating the plant growth response under saline conditions via the regulation of the stomata response to stress. This role seems to be independent of the RLCK311 kinase activity, since the overexpression of the RLCK311 C-terminus (ΔN), which lacks the kinase full domain, has a similar phenotype to RLCK311FL.

Tomato yellow leaf curl virus (TYLCV)-resistant tomatoes share molecular mechanisms sustaining resistance with their wild progenitor Solanum habrochaites but not with TYLCV-susceptible tomatoes.

The wild tomato species Solanum habrochaites (Sh) has been used as a source for tomato yellow leaf curl virus (TYLCV) resistance in a breeding program to generate a TYLCV-resistant tomato line. Susceptible (S) and resistant (R) lines have been developed through this program. We compared the behavior of R, S and Sh tomato plants upon infection to find out whether the resistant phenotype of R plants originated from Sh. Results showed that mechanisms involving sugar-signaling (i.e., LIN6/HT1), water channels (i.e., TIP1;1), hormone homeostasis (i.e., ABA and SA) and urea accumulation were shared by S. habrochaites and R plants, but not by S. habrochaites and S tomatoes. This finding supports the hypothesis that these mechanisms were introgressed in the R genotype from the wild tomato progenitor during breeding for TYLCV resistance. Hence, identification of genes contributing to resistance to biotic stress from wild tomato species and their introgression into domestic plants ensures tomato supply and food security.

Measuring the Hydraulic Conductivity of Grass Root Systems.

Root-system hydraulic conductivity (RSHC) is an important physiological characteristic that describes the inherent ability of roots to conduct water across a water-potential gradient between the root and the stem xylem. RSHC is commonly used as an indicator of plant functioning and adaptability to a given environment. A simple, fast, and easy-to-use protocol is described for the quantification of RSHC at the seedling stage in two important monocot species grown in hydroponic solution: Setaria viridis, a C4 model plant, and wheat, a C3 crop plant. This protocol can also be easily modified for use with almost any grass species and environmental treatments, such as salinity or hormone treatments. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Setaria hydrostatic root-system hydraulic conductivity Alternate Protocol: Measuring the root conductivity of young plants with soft stems.

Correlation-based network analysis combined with machine learning techniques highlight the role of the GABA shunt in Brachypodium sylvaticum freezing tolerance.

Perennial grasses will account for approximately 16 billion gallons of renewable fuels by the year 2022, contributing significantly to carbon and nitrogen sequestration. However, perennial grasses productivity can be limited by severe freezing conditions in some geographical areas, although these risks could decrease with the advance of climate warming, the possibility of unpredictable early cold events cannot be discarded. We conducted a study on the model perennial grass Brachypodium sylvaticum to investigate the molecular mechanisms that contribute to cold and freezing adaption. The study was performed on two different B. sylvaticum accessions, Ain1 and Osl1, typical to warm and cold climates, respectively. Both accessions were grown under controlled conditions with subsequent cold acclimation followed by freezing stress. For each treatment a set of morphological parameters, transcription, metabolite, and lipid profiles were measured. State-of-the-art algorithms were employed to analyze cross-component relationships. Phenotypic analysis revealed higher adaption of Osl1 to freezing stress. Our analysis highlighted the differential regulation of the TCA cycle and the GABA shunt between Ain1 and Osl1. Osl1 adapted to freezing stress by repressing the GABA shunt activity, avoiding the detrimental reduction in fatty acid biosynthesis and the concomitant detrimental effects on membrane integrity.

Silencing of OsCV (chloroplast vesiculation) maintained photorespiration and N assimilation in rice plants grown under elevated CO2.

High CO2 concentrations stimulate net photosynthesis by increasing CO2 substrate availability for Rubisco, simultaneously suppressing photorespiration. Previously, we reported that silencing the chloroplast vesiculation (cv) gene in rice increased source fitness, through the maintenance of chloroplast stability and the expression of photorespiration-associated genes. Because high atmospheric CO2 conditions diminished photorespiration, we tested whether CV silencing might be a viable strategy to improve the effects of high CO2 on grain yield and N assimilation in rice. Under elevated CO2 , OsCV expression was induced, and OsCV was targeted to peroxisomes where it facilitated the removal of OsPEX11-1 from the peroxisome and delivered it to the vacuole for degradation. This process correlated well with the reduction in the number of peroxisomes, the decreased catalase activity and the increased H2 O2 content in wild-type plants under elevated CO2 . At elevated CO2 , CV-silenced rice plants maintained peroxisome proliferation and photorespiration and displayed higher N assimilation than wild-type plants. This was supported by higher activity of enzymes involved in NO3- and NH4+ assimilation and higher total and seed protein contents. Co-immunoprecipitation of OsCV-interacting proteins suggested that, similar to its role in chloroplast protein turnover, OsCV acted as a scaffold, binding peroxisomal proteins.