RESUMO
The LATE EMBRYOGENESIS ABUNDANT PROTEINs (LEAPs) are a class of noncatalytic, intrinsically disordered proteins with a malleable structure. Some LEAPs exhibit a protein and/or membrane binding capacity and LEAP binding to various targets has been positively correlated with abiotic stress tolerance. Regarding the LEAPs' presumptive role in protein protection, identifying client proteins (CtPs) to which LEAPs bind is one practicable means of revealing the mechanism by which they exert their function. To this end, we used phage display affinity selection to screen libraries derived from Arabidopsis thaliana seed mRNA with recombinant orthologous LEAPs from Arabidopsis and soybean (Glycine max). Subsequent high throughput sequencing of DNA from affinity-purified phage was performed to characterize the entire sub-population of phage retained by each LEAP orthologue. This entailed cataloging in-frame fusions, elimination of false positives, and aligning the hits on the CtP scaffold to reveal domains of respective CtPs that bound to orthologous LEAPs. This approach (Paired-end PhAge Sequencing, or PEPA-Seq) revealed a subpopulation of the proteome constituting the CtP repertoire in common between the two DHNs orthologues (LEA14 and GmPm12) compared to BSA (unrelated binding control). The veracity of LEAP:CtP binding for one of the CtPs (LEA14 and GmPM12 self-association) was independently assessed using temperature related intensity change (TRIC) analysis. Moreover, LEAP:CtP interactions for four other CtPs were confirmed in planta using bimolecular fluorescence complementation (BiFC) assays. The results provide insights into the involvement of the DHN Y-segments and K-domains in protein binding.
RESUMO
The cold stress susceptibility of tomato (Solanum lycopersicum) curtails its cultivation, with significant impact in temperate regions and on cropping seasons. To unravel genomic regions responsible for cold stress resilience, a diverse set of fifty genotypes encompassing cultivated, wild species, and landraces were genotyped using genotyping-by-sequencing. Over two years and six trials employing both early and late sowing, these lines were evaluated. Illumina-based next-generation sequencing produced up to 3 million reads per sample from individually sequenced library pools. The Tassel pipeline yielded 10,802 variants, subsequently filtered to 3,854 SNPs for genome-wide association analysis (GWAS). Employing clustering methods (population structure) via TASSEL, SNPhylo, and Kinship matrix, the fifty genotypes clustered into four distinct gene pools. The GWAS for cold tolerance in tomato integrated key traits including yield. Using six independent phenotypic datasets representing various environments, the study identified 4,517 significant marker-trait associations for cold tolerance traits. Notably, pivotal variations (> 10%) in cold stress tolerance, particularly proline content, were linked to marker-trait associations. Additionally, 5,727 significant marker-trait associations for yield and yield-related traits were unveiled, shedding light on fruit yield and directly associated attributes. The investigation pinpointed 685 candidate genes across all examined traits, including 60 genes associated with biological processes within these genomic regions. Remarkably, 7 out of the 60 genes were directly linked to abiotic stress tolerance, functioning as stress-responsive genes either directly or indirectly. The identified genes, particularly those associated with stress response, could hold the key to enhancing cold tolerance and overall crop productivity in tomato cultivation.