Subcellular Localization of Seed-Expressed LEA_4 Proteins Reveals Liquid-Liquid Phase Separation for LEA9 and for LEA48 Homo- and LEA42-LEA48 Heterodimers

Abstract

LEA proteins are involved in plant stress tolerance. In Arabidopsis, the LEA_4 Pfam group is the biggest group with the majority of its members being expressed in dry seeds. To assess subcellular localization in vivo, we investigated 11 seed-expressed LEA_4 proteins in embryos dissected from dry seeds expressing LEA_4 fusion proteins under its native promoters with the Venus fluorescent protein (proLEA_4::LEA_4:Venus). LEA_4 proteins were shown to be localized in the endoplasmic reticulum, nucleus, mitochondria, and plastids. LEA9, in addition to the nucleus, was also found in cytoplasmic condensates in dry seeds dependent on cellular hydration level. Most investigated LEA_4 proteins were detected in 4-d-old seedlings. In addition, we assessed bioinformatic tools for predicting subcellular localization and promoter motifs of 11 seed-expressed LEA_4 proteins. Ratiometric bimolecular fluorescence complementation assays showed that LEA7, LEA29, and LEA48 form homodimers while heterodimers were formed between LEA7-LEA29 and LEA42-LEA48 in tobacco leaves. Interestingly, LEA48 homodimers and LEA42-LEA48 heterodimers formed droplets structures with liquid-like behavior. These structures, along with LEA9 cytoplasmic condensates, may have been formed through liquid-liquid phase separation. These findings suggest possible important roles of LLPS for LEA protein functions.

In Silico Characterisation of the Late Embryogenesis Abundant (LEA) Protein Families and Their Role in Desiccation Tolerance in Ramonda serbica Panc

“…However, in the presence of a membrane surface, dehydrins can gain a partial helical structure [ 72 ]. The representative RsLEA_139 ( Figure 5 ) dehydrin was predicted to be highly disordered in the solution (97%), suggesting its possible involvement in liquid–liquid phase separation (LLPS), followed by proteinaceous condensates formation, similar to how it was recently experimentally confirmed for two Arabidopsis LEAPs predicted to be 100% disordered [ 35 ]. Therefore, nuclear desiccation-inducible RsLEA_139 might be involved in the LLPS-related dynamic assembly of nuclear compartments such as nuclear bodies and chromatin structures [ 73 ] as a part of gene expression regulation during desiccation in R. serbica .…”

“…Most LEAPs are predicted to be intrinsically disordered proteins (IDPs) [ 31 , 35 ]. The flexible structure of IDPs imposes restrictions on their 3D structure determination, as can be evidenced by a low number of deposed IDPs in the Protein Data Bank (PDB) [ 58 ].…”

“…At this moment, no specific physiological function was attributed to LEAPs [ 3 , 35 ]. Their high structural plasticity allows them to interact with various ligands and partners.…”

“…However, the details regarding the recruitment of specific or nonspecific target/client proteins and the importance of physical separation within the proteinaceous condensates among various cellular components are still debatable [ 3 ]. The influence of the microenvironment (i.e., pH, osmotic potential, and ionic strength); presence of other solutes (sucrose and raffinose); and posttranslational modifications on specific LEAP disorder-to-order transitions and, therefore, functions are still elusive [ 35 ].…”

Proteins Involved in Plant Dehydration Protection: The Late Embryogenesis Abundant Family

“…These proteins are able to weakly associate with themselves or RNA, resulting in the formation of a membraneless organelle. Ginsawaeng et al [ 5 ] studied several LEA proteins from Arabidopsis thaliana using a combination of bioinformatics and fluorescence microscopy to examine the localization and phase separation properties of these proteins. In terms of self-association, they found that some LEA proteins could form oligomers, and, most interestingly, LEA proteins LEA9 and LEA48 formed droplet structures, showing that they form LLPS condensates.…”