Redox-engineering enhances maize thermotolerance and grain yield in the field

Contains fulltext : 252904.pdf (Publisher’s version ) (Open Access)08 juli 202

Tomato class II glutaredoxin mutants generated via multiplex CRISPR/Cas9 genome editing technology are susceptible to multiple abiotic stresses

Doctor of PhilosophyDepartment of Horticulture and Natural ResourcesSunghun ParkGene silencing technologies such as clustered regulatory short palindromic repeats (CRISPR)/Cas9 and RNA interference (RNAi) created a revolution in genome engineering. They are highly site-specific, simple, fast, and cost-effective. Since their discovery, gene silencing technologies have extensively been implemented in various organisms including humans, animals, plants, and microbes. They have been used for both basic science studies such as gene functional analysis and applied science such as medicine and crop improvement. In this work, we used multiplex CRISPR/Cas9 system to knock out all four members of the class II glutaredoxin (GRX) gene family including S14, S15, S16 and S17 in tomato and RNAi technology to express mouse complement 3 (C3) and complement factor 7 (CF7) small interfering RNAs (siRNAs) in lettuce. Reactive oxygen species (ROS) are induced under abiotic and biotic stresses and also as byproducts of aerobic metabolism, and their overproduction causes oxidative damage to macromolecules such as proteins, lipids, carbohydrates, and nucleic acids. GRXs are small ubiquitous proteins that are known to be involved in cellular redox homeostasis by reducing disulfate bonds and scavenging ROS. To investigate the functions of each member of class II GRX gene family in tomato’s response to abiotic stresses, single and multiple knockout lines for class II GRXs were obtained using multiplex CRISPR/Cas9 system. Mutant lines and wild-type plants were subjected to heat, drought, chilling, cadmium (Cd) toxicity, and short photoperiod stresses. Phenotyping data showed that members of the class II GRX gene family are critical for tomato’s growth, development, and survival under several abiotic stresses. Our findings propose novel functions for members of class II SlGRXs. RNAi technology can be utilized to target disease-related proteins. However, the application of siRNAs is challenging predominantly due to the difficult delivery and instability of siRNA into the host system. Recent findings of bioactivity and bioavailability of plants’ miRNAs through animals’ digestive systems led to the newly introduced field of dietary siRNAs. Animal’s siRNA can be expressed in plant tissues and delivered as dietary siRNA. Here, an expression vector made based on a rice miRNA backbone, Osa-MIR528, was utilized to construct two plant expression vectors containing siRNAs silencing mouse C3 and CF7 proteins. Both C3 and CF7 proteins are involved in blood clotting which could lead to cardiovascular dysfunction. Expression of both primary and mature C3 and CF7 siRNAs in lettuce was validated by semi quantitative real-time PCR and end-point PCR, respectively, and was confirmed via Sanger sequencing. Established amiRNA system in lettuce through this work will have further applications. As an edible leafy plant with high biomass, lettuce can be used as a valuable host to produce various diseases targeting siRNAs

Expression analyses of salinity stress- associated ESTs in Aeluropus littoralis

Salinity is among the most important abiotic stresses affecting crop production throughout the earth. Halophyte plants can sustain high salinity levels, therefore elucidating molecular mechanisms underlying their salinity resistance is beneficial for crop improvement. Aeluropus littoralis, a halophyte weed, is a great genetic resource for this purpose. Isolated expressed sequence taq (EST) sequences from A. littoralis under salinity stress, have given us the chance to find and analyze transcripts of genes involved in response to salinity. Transcriptome analyses indicated the expression levels of mRNAs corresponding to 10 of sequences were increased under treatments. All mRNAs were significantly induced under salt treatment with the highest peaks observed at different hours of treatments. Moreover, the full-length cDNA of vacuolar H+-pyrophosphatase (VP) was isolated utilizing 3′ and 5′ rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) and characterized (GenBank accession number of KT253223.1). The extracted full-length of VP was 2732 bp, which contained ORF of 2292 bp encoding 763 amino acids.</p

Ectopic Expression of a Heterologous Glutaredoxin Enhances Drought Tolerance and Grain Yield in Field Grown Maize

Drought stress is a major constraint in global maize production, causing almost 30–90% of the yield loss depending upon growth stage and the degree and duration of the stress. Here, we report that ectopic expression of Arabidopsis glutaredoxin S17 (AtGRXS17) in field grown maize conferred tolerance to drought stress during the reproductive stage, which is the most drought sensitive stage for seed set and, consequently, grain yield. AtGRXS17-expressing maize lines displayed higher seed set in the field, resulting in 2-fold and 1.5-fold increase in yield in comparison to the non-transgenic plants when challenged with drought stress at the tasseling and silking/pollination stages, respectively. AtGRXS17-expressing lines showed higher relative water content, higher chlorophyll content, and less hydrogen peroxide accumulation than wild-type (WT) control plants under drought conditions. AtGRXS17-expressing lines also exhibited at least 2-fold more pollen germination than WT plants under drought stress. Compared to the transgenic maize, WT controls accumulated higher amount of proline, indicating that WT plants were more stressed over the same period. The results present a robust and simple strategy for meeting rising yield demands in maize under water limiting conditions

Redox-engineering enhances maize thermotolerance and grain yield in the field

Increasing populations and temperatures are expected to escalate food demands beyond production capacities, and the development of maize lines with better performance under heat stress is desirable. Here, we report that constitutive ectopic expression of a heterologous glutaredoxin S17 from Arabidopsis thaliana (AtGRXS17) can provide thermotolerance in maize through enhanced chaperone activity and modulation of heat stress-associated gene expression. The thermotolerant maize lines had increased protection against protein damage and yielded a 6-fold increase in grain production in comparison to the non-transgenic counterparts under heat stress field conditions. The maize lines also displayed thermotolerance in the reproductive stages, resulting in improved pollen germination and the higher fidelity of fertilized ovules under heat stress conditions. Our results present a robust and simple strategy for meeting rising yield demands in maize and, possibly, other crop species in a warming global environment