Abstract
Although plants as sessile organisms are affected by a variety of stressors in the field, the stress factors for the above-ground and underground parts of the plant and their gene expression profiles are not the same. Here, we investigated NbKPILP, a gene encoding a new member of the ubiquitous, pathogenesis-related Kunitz peptidase inhibitor (KPI)-like protein family, that we discovered in the genome of Nicotiana benthamiana and other representatives of the Solanaceae family. The NbKPILP gene encodes a protein that has all the structural elements characteristic of KPI but in contrast to the proven A. thaliana KPI (AtKPI), it does not inhibit serine peptidases. Unlike roots, NbKPILP mRNA and its corresponding protein were not detected in intact leaves, but abiotic and biotic stressors drastically affected NbKPILP mRNA accumulation. In search of the causes of suppressed NbKPILP mRNA accumulation in leaves, we found that the NbKPILP gene is “matryoshka,” containing an alternative nested reading frame (ANRF) encoding a 53-amino acid (aa) polypeptide (53aa-ANRF) which has an amphipathic helix (AH). We confirmed ANRF expression experimentally. A vector containing a GFP-encoding sequence was inserted into the NbKPILP gene in frame with 53aa-ANRF, resulting in a 53aa-GFP fused protein that localized in the membrane fraction of cells. Using the 5′-RACE approach, we have shown that the expression of ANRF was not explained by the existence of a cryptic promoter within the NbKPILP gene but was controlled by the maternal NbKPILP mRNA. We found that insertion of mutations destroying the 53aa-ANRF AH resulted in more than a two-fold increase of the NbKPILP mRNA level. The NbKPILP gene represents the first example of ANRF functioning as a repressor of a maternal gene in an intact plant. We proposed a model where the stress influencing the translation initiation promotes the accumulation of NbKPILP and its mRNA in leaves.
Highlights
We found that the Nicotiana benthamiana gene encoding a KPI-like protein (NbKPILP) gene is common for Solanaceae plants and belongs to a matryoshka gene family containing an alternative nested reading frame (ANRF) that encodes a 53-amino acid polypeptide (53aa-ANRF) which contains an amphipathic helix (AH)
In a study of the transcriptome of N. benthamiana leaves infected with Tobacco mosaic virus (TMV), we detected an increased accumulation of mRNA (EMBL ID FN687760) encoding KPIlike protein (NbKPILP) (NCBI accession number D4IHB9), which according to the MEROPS classification
The genomic fragment sequence perfectly matched the cDNA-derived fragment, which indicated the absence of an intron in the ORF-encoding part of chromosomal NbKPILP gene
Summary
Plants in natural field conditions are constantly exposed to a variety of abiotic and biotic environmental factors (Wang et al, 2003; Chaves and Oliveira, 2004; Nakashima and YamaguchiShinozaki, 2006; Hirel et al, 2007; Bailey-Serres and Voesenek, 2008; Atkinson and Urwin, 2012; Suzuki et al, 2014; Ramegowda and Senthil-Kumar, 2015).A set of stress factors and, gene expression profiles for the underground and above-ground parts of plants differ (Freschet et al, 2015; Islam et al, 2015a,b; Liu et al, 2015; Chmielewska et al, 2016). The plant, in response to the impact of a stress factor, switches on protective mechanisms, including generalized cellular mobilization and accumulation of mRNAs that direct the synthesis of protective proteins in cells (Dowen et al, 2012; Zavaliev et al, 2013; Crisp et al, 2016; Yang et al, 2017; Zhang and Sonnewald, 2017). Termination of the stress-factor impact cancels the synthesis of protective protein mRNAs and is followed by their degradation. Such repeated effects create a kind of “memory” (Crisp et al, 2016), which allows plants to shorten the time for a protective response. The translation of mRNA is a mechanism for responding to stress, especially the stages involving the 5′ terminal oligopyrimidine tract and translation initiation factors eIF4E and eIF2a (Sesma et al, 2017)
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