WHIRLY1 Interactome Profiling Reveals the Chloroplast Fate Correlation with Stromule-like Morphologies in Arabidopsis Leaves Under Dark and DBMIB Stress Conditions

The WHIRLY family comprises DNA/RNA-binding proteins localized to multiple subcellular compartments, including organelles and the nucleus. However, the subcellular distribution and functional roles of WHIRLY protein complexes are not fully characterized. In this study, a WHIRLY1 protein pulldown coupled with mass spectrometry analysis was used to profile the interacting proteome in an Arabidopsis thaliana plant overexpressing WHIRLY1–GFP. This analysis identified over 770 interacting proteins, with RNA-binding proteins representing the most enriched functional category. Subcellular compartmental mapping revealed distinct spatial organization: oxidation-related, cytoskeleton-associated, and chloroplast/mitochondrion-targeted RNA-binding proteins were significantly enriched at the chloroplast periphery. Conversely, a subset of nuclear envelope-associated proteins, including NOT3, TUBB3, and histone H4, were connected with the formation of tubular stromule-like structures under growth light conditions as opposed to extended dark conditions. Under DBMIB-induced stress, however, reconfiguration of the WHIRLY1 interactome occurred: plastid ribosomal structural components declined, while the transcription factor GRF6 accumulated in the nucleus, concomitant with the formation of vesicular stromule-like structures. Biomolecular fluorescence complementation and yeast two-hybrid assays confirmed direct interactions between WHIRLY1 and stromule-localized proteins, several RNA-binding proteins, and nuclear proteins. Collectively, these findings suggest that the dynamic assembly of the WHIRLY1 interactome in conjunction with stromule biogenesis acts as a regulatory nexus coordinating chloroplast functions under stressful conditions.