Photorespiration metabolizes 2-phosphoglyolate (2-PG) to avoid inhibition of carbon assimilation and allocation. In addition to 2-PG removal, photorespiration has been shown to play a role in stress protection. Here, we studied the impact of faster 2-PG degradation through overexpression of 2-PG phosphatase (PGLP) on the abiotic stress-response of Arabidopsis thaliana (Arabidopsis). Two transgenic lines and the wild type were subjected to short-time high light and elevated temperature stress during gas exchange measurements. Furthermore, the same lines were exposed to long-term water shortage and elevated temperature stresses. Faster 2-PG degradation allowed maintenance of photosynthesis at combined light and temperatures stress and under water-limiting conditions. The PGLP-overexpressing lines also showed higher photosynthesis compared to the wild type if grown in high temperatures, which also led to increased starch accumulation and shifts in soluble sugar contents. However, only minor effects were detected on amino and organic acid levels. The wild type responded to elevated temperatures with elevated mRNA and protein levels of photorespiratory enzymes, while the transgenic lines displayed only minor changes. Collectively, these results strengthen our previous hypothesis that a faster photorespiratory metabolism improves tolerance against unfavorable environmental conditions, such as high light intensity and temperature as well as drought. In case of PGLP, the likely mechanism is alleviation of inhibitory feedback of 2-PG onto the Calvin–Benson cycle, facilitating carbon assimilation and accumulation of transitory starch.

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.

During seed germination, desiccation tolerance is lost in the radicle with progressing radicle protrusion and seedling establishment. This process is accompanied by comprehensive changes in the metabolome and proteome. Germination of Arabidopsis seeds was investigated over 72 h with special focus on the heat-stable proteome including late embryogenesis abundant (LEA) proteins together with changes in primary metabolites. Six metabolites in dry seeds known to be important for seed longevity decreased during germination and seedling establishment, while all other metabolites increased simultaneously with activation of growth and development. Thermo-stable proteins were associated with a multitude of biological processes. In the heat-stable proteome, a relatively similar proportion of fully ordered and fully intrinsically disordered proteins (IDP) was discovered. Highly disordered proteins were found to be associated with functional categories development, protein, RNA and stress. As expected, the majority of LEA proteins decreased during germination and seedling establishment. However, four germination-specific dehydrins were identified, not present in dry seeds. A network analysis of proteins, metabolites and amino acids generated during the course of germination revealed a highly connected LEA protein network.