Biology subjects

Karavolias, N. G.

Publications and source records attributed to Karavolias, N. G..

4 recordsLinked to original sources

bioRxiv2023

Editing cis-regulatory elements towards generating rice stomatal morphological variation for adaptation to broad and dynamic environments

Cis-regulatory element editing can generate quantitative trait variation while mitigating against extreme phenotypes and harmful pleiotropy associated with coding sequence mutations. Here, we applied a multiplexed guide RNA design approach, informed by bioinformatic datasets, to generate genotypic variation in the promoter of OsSTOMAGEN, a positive regulator of stomatal density in rice. Engineered genotypic variation corresponded to broad and continuous variation in stomatal density, ranging from 70% to 120% of wild-type stomatal density. This near-isogenic panel of stomatal variants was leveraged in physiological assays to establish discrete relationships between stomatal morphological variation and stomatal conductance, carbon assimilation, and intrinsic water use efficiency in steady-state and fluctuating light conditions. Additionally, promoter alleles were subjected to vegetative drought regimes to assay the effects of the edited alleles on developmental response to drought. Notably, the capacity for drought-responsive stomatal density reprogramming in stomagen and two cis-regulatory edited alleles was reduced. Collectively our data demonstrate that cis-regulatory element editing can generate near-isogenic trait variation that can be leveraged for establishing relationships between anatomy, physiology, and crop improvement along diverse environmental clines.

plant biology↗

bioRxiv2023

Multiplexed CRISPR/Cas9 mutagenesis of rice PSBS1 non-coding sequences for transgene-free overexpression

Understanding CRISPR/Cas9s capacity to generate native overexpression (OX) alleles would accelerate agronomic gains achievable by gene editing. To generate OX alleles with increased RNA and protein abundance, we leveraged multiplexed CRISPR/Cas9 mutagenesis of non-coding DNA sequences located upstream of the rice PSBS1 gene. We isolated 120 transgene-free, gene-edited alleles with varying NPQ capacity in vivo --ranging from complete knockout to overexpression, using a high-throughput phenotyping and transgene screening pipeline. Overexpression of OsPSBS1 increased protein abundance 2-3-fold, matching fold changes obtained by transgenesis. Increased PsbS protein abundance enhanced non-photochemical quenching capacity and improved water-use efficiency. Across our resolved genetic variation, we identify the role of 5UTR indels and inversions in driving knockout/knockdown and overexpression phenotypes, respectively. Complex structural variants, such as the 252kb duplication/inversion generated in this study, evidence the potential of CRISPR/Cas9 to facilitate significant genomic changes with negligible off-target transcriptomic perturbations. Our results may inform future gene-editing strategies for hypermorphic alleles and have opened the door to the pursuit of gene-edited, non-transgenic rice plants with accelerated relaxation of photoprotection.

plant biology↗

bioRxiv2023

Chlorophyll to Zeaxanthin Energy Transfer in Non-Photochemical Quenching: An Exciton Annihilation-free Transient Absorption Study

Zeaxanthin (Zea) is a key component in the energy-dependent, rapidly reversible, non-photochemical quenching process (qE) that regulates photosynthetic light harvesting. Previous transient absorption (TA) studies suggested that Zea can participate in direct quenching via Chlorophyll (Chl) to Zea energy transfer. However, the contamination of intrinsic exciton-exciton annihilation (EEA) makes the assignment of TA signal ambiguous. In this study, we present EEA-free TA data using Nicotiana benthamiana thylakoid membranes, including wild type and three NPQ mutants (npq1, npq4, and lut2) generated by CRISPR/Cas9 mutagenesis. Results show a strong correlation between excitation energy transfer from excited Chl Qy to Zea S1 and the xanthophyll cycle during qE activation. Notably, a Lut S1 signal is absent in the npq1 thylakoids which lack zeaxanthin. Additionally, the fifth-order response analysis shows a reduction in the exciton diffusion length (LD) from 55 {+/-} 5 nm to 38 {+/-} 3 nm under high light illumination, consistent with the reduced range of exciton motion being a key aspect of plants response to excess light.

plant biology↗

bioRxiv2019

Low additive genetic variation in a trait under selection in domesticated rice

Quantitative traits are important targets of both natural and artificial selection. The genetic architecture of these traits and its change during the adaptive process is thus of fundamental interest. The fate of the additive effects of variants underlying a trait receives particular attention because they constitute the genetic variation component that is transferred from parents to offspring and thus governs the response to selection. While estimation of this component of phenotypic variation is challenging, the increasing availability of dense molecular markers puts it within reach. Inbred plant species offer an additional advantage because phenotypes of genetically identical individuals can be measured in replicate. This makes it possible to estimate marker effects separately from the contribution of the genetic background. We focused on root growth in domesticated rice, Oryza sativa, under normal and aluminum (Al) stress conditions, a trait under recent selection because it correlates with survival under drought. A dense single nucleotide polymorphism (SNP) map is available for all accessions studied. Taking advantage of this map and a set of Bayesian models, we assessed additive marker effects. While total genetic variation accounted for a large proportion of phenotypic variance, marker effects contributed little information, particularly in the Al-tolerant tropical japonica population of rice. We were unable to identify any loci associated with root growth in this population. Models estimating the aggregate effects of all measured genotypes like-wise produced low estimates of marker heritability and were unable to predict total genetic values accurately. Our results support the long-standing conjecture that additive genetic variation is depleted in traits under selection. We further provide evidence that this depletion is due to the prevalence of low-frequency alleles that underlie the trait.

genetics↗