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.

中文翻译：

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种子表达的 LEA_4 蛋白的亚细胞定位揭示了 LEA9 和 LEA48 同源二聚体和 LEA42-LEA48 异二聚体的液液相分离

LEA 蛋白参与植物胁迫耐受性。在拟南芥中，LEA_4 Pfam 组是最大的组，其大多数成员在干种子中表达。为了评估体内亚细胞定位，我们研究了 11 种种子表达的 LEA_4 蛋白，这些蛋白是从干燥种子中分离出来的，在其天然启动子下用金星荧光蛋白 ( proLEA_4::LEA_4:Venus)表达 LEA_4 融合蛋白。）。LEA_4 蛋白显示在内质网、细胞核、线粒体和质体中定位。除了细胞核外，LEA9 也存在于干燥种子的细胞质凝聚物中，这取决于细胞水合水平。大多数研究的 LEA_4 蛋白是在 4 天龄幼苗中检测到的。此外，我们评估了用于预测 11 种种子表达的 LEA_4 蛋白的亚细胞定位和启动子基序的生物信息学工具。比率双分子荧光互补分析表明，LEA7、LEA29 和 LEA48 在烟叶中形成同源二聚体，而异源二聚体则在 LEA7-LEA29 和 LEA42-LEA48 之间形成。有趣的是，LEA48 同源二聚体和 LEA42-LEA48 异源二聚体形成了具有类似液体行为的液滴结构。这些结构，连同 LEA9 细胞质凝聚物，可能是通过液-液相分离形成的。这些发现表明 LLPS 可能对 LEA 蛋白功能发挥重要作用。