AtQQS orphan gene and NtNF-YC4 boost protein accumulation and pest resistance in tobacco (Nicotiana tabacum).

Qua-Quine Starch (QQS), an orphan gene exclusively found in Arabidopsis thaliana, interacts with Nuclear Factor Y subunit C4 (NF-YC4) and regulates carbon and nitrogen allocation in different plant species. Several studies uncovered its potential in increasing total protein and resistance against pathogens/pests in Arabidopsis and soybean. However, it is still unclear if these attributes QQS offers are universal in all flowering plants. Here we studied AtQQS and Nicotiana tabacum NF-YC4's (NtNF-YC4) influence on starch/protein content and pest resistance in tobacco. Our results showed both AtQQS and NtNF-YC4 had a positive impact on the plant's total protein accumulation. Simultaneously, we have also observed reduced starch biosynthesis and increased resistance against common pests like whiteflies (Bemisia tabaci) and aphids (Myzus persicae) in tobacco plants expressing AtQQS or overexpressing NtNF-YC4. Real-time PCR also revealed increased NF-YC4 expression after aphid infestation in tobacco varieties with higher pest resistance but decreased/unchanged NF-YC4 expression in varieties susceptible to pests. Further analysis revealed that QQS expression and overexpression of NtNF-YC4 strongly repressed expression of genes such as sugar transporter SWEET10 and Flowering Locus T (FT), suggesting involvement of SWEET10 and FT in the QQS and NF-YC4 mediated carbon and nitrogen allocation in tobacco. Our data suggested that the activity of species-specific orphan genes may not be limited to the original species or its close relatives. Sequence alignment revealed the conserved sequence of the NF-YC4s in different plant species that may be responsible for the resulting shift in metabolism, pest resistance. Cis-acting DNA element analysis of NtNF-YC4 promoter region may outline potential mechanisms for these phenotypic changes.