Functional characterization of orphan genes in Arabidopsis thaliana

All organisms are known to contain a unique set of genes called 'orphan genes', which do not share any similarity with genes or protein domains in other evolutionary lineages. Such orphan genes can impart unique traits to the organism resulting in lineage specific evolutionary innovation. In this work Brassicaceae specific orphan genes in Arabidopsis thaliana were analysed for their functionality in abiotic stress responses. Abiotic stress responsive orphan genes were identified based on their differential expression under various stress conditions and were functionally screened using three different approaches; loss-of-function screen, gain-of-function screen and gain-of-function screen by heterologous expression. From the loss of function screen, one orphan gene (ASR50) was identified which confers tolerance to salt stress when knocked out. The mutant asr50-1 also shows high photosynthetic efficiency under salt stress compared to wild type. The gain-of-function screen in A. thaliana resulted in two promising candidates, ASR35 and ASR63, which conferred tolerance to salt stress and iron deficiency respectively. The third approach, a gain-of-function screen by heterologous expression using yeast stress sensitive mutants, identified a total of 27 orphan genes conferring varied degrees of stress tolerance or sensitivity to different abiotic stress conditions. Importantly, this approach also indicated that orphan gene functionality is transferable to other lineages. The overexpression lines of ASR63 (35S::ASR63) were further analysed to identify the possible mechanism of tolerance conferred under iron deficiency. The results suggested that the 35S::ASR63 lines accumulated more iron in shoots and in the mature seeds compared to wild type control. Furthermore, the transgenic lines were found to produce higher levels of the antioxidants glutathione and ascorbic acid in the shoots which possibly maintains favourable redox balance under iron deficiency for the normal uptake of iron. In addition, the seeds of 35S::ASR63 lines show germination sensitivity to ABA and the GA inhibitor PAC which needs further characterization. Our hypothesis is that ASR63 is a de novo gene originated due to the divergent transcription of a 'bifunctional' promoter of an upstream gene. Overall, this study highlights the importance of orphan genes and adds to the increasing evidences for functionality of orphan genes giving rise to evolutionary innovations within lineages.2021-08-3