Evaluation of drought tolerance in genetically modified Lolium perenne L. and Arabidopsis thaliana

Abstract

Ubiquitin like proteins (UBLs) are known to have different functions including a potential role in drought tolerance of perennial ryegrass (Lolium perenne L.). UBLs are related in tertiary structure to ubiquitin (UB) and they both serve as regulators of many cellular processes. In previous work over-expression of one UBL, the Lolium perenne L. Ubl5 (LpUbl5) homologue in perennial ryegrass resulted in plants with enhanced drought tolerance. Other studies in the nematode Caenorhabditis elegans have associated Ubl5 with a mitochondrial unfolded protein response (UPRmt), and studies in yeast and human cells suggest a role in alternative splicing for Ubl5. This PhD study aimed to characterize the LpUbl5 gene in the context of its potential role in drought tolerance using LpUbl5-overexpressing perennial ryegrass. The study extended to include Arabidopsis thaliana transformed using LpUbl5 to support the findings. Subsequently subcellular localization of LpUBL5 protein was investigated to identify its potential role in alternative splicing. The study also screened different perennial ryegrass accessions from diverse backgrounds to identify germplasm with potential tolerance under drought stress. The results rejected any role of the LpUbl5 gene in drought tolerance of perennial ryegrass and A. thaliana. A potential seed lethal phenotype was observed in Ubl5 mutants of A. thaliana. The study found co-localization of the LpUBL5 protein into the cytoplasm and nucleus of Nicotiana benthamiana (tobacco) leaves and Allium cepa L. (onion) cells. This supports its potential role in alternative splicing. Importantly, the study also identified a germplasm accession of Lolium perenne L. from Norway with increased drought tolerance in a controlled environment. The results generated from the LpUbl5 overexpressing perennial ryegrass lines emphasize the need to integrate critical evaluation of plant lines in their developmental stages especially in out crossing species. Lack of physiological evidence rejected the potential role of LpUbl5 in drought tolerance. However, the localization of LpUBL5 indicates its functional significance as UBL5 homologues from vertebrates, yeasts and plants and exhibits high levels of protein sequence conservation. It is conceivable that UBL5 homologues possess similar functions across species. The indications of a potential seed-lethal phenotype of mutants is consistent with findings in humans, yeast and C.elegans. Future research is recommended to explore and confirm the potential seed lethal phenotype of Ubl5 mutation in plants using Arabidopsis. This may identify the phenotype characters of Ubl5 in plants. Since cultivar development is an integral part of pasture development, the genetic diversity of Norwegian accession provides a candidate to develop perennial ryegrass plants with enhanced drought tolerance.