Biology subjects

Pulido, P.

Publications and source records attributed to Pulido, P..

3 recordsLinked to original sources

bioRxiv2025

CLPC2 plays specific roles in CLP complex-mediated regulation of growth, photosynthesis, embryogenesis and response to growth-promoting microbial compounds

In Arabidopsis, exposure to growth-promoting microbial volatile compounds (VCs) enhances CLPC2 levels. This chaperone forms part of the CLP protease complex, which ensures the correct functioning of essential processes in plastids. Previous studies indicated considerable functional redundancy of CLPC2 with its dominant paralogue CLPC1. However, the function and action mechanism of CLPC2 still remain unknown. Here we found that CLPC2-lacking clpc2-2 mutants were unresponsive to microbial VCs, whereas clpc1-1 knockout mutants exhibited a WT-like response to VCs when grown on sucrose-containing medium. Unlike clpc1-1, clpc2-2 plants presented a fully functional photosystem II and lower than WT stomatal conductance. Furthermore, clpc2-2 plants, but not clpc1-1 plants, produced wrinkled seeds with delayed embryonic development and reduced postgerminative establishment rates that resembled those of mutants lacking P and R components of the CLP proteolytic core. Proteomic analyses revealed that knocking out of CLPC2 enhanced the levels of chloroplastic proteins that are essential for growth, embryo development and seedling establishment. These changes differed from those promoted by the lack of CLPC1, but partially resembled those promoted by CLPPR core inactivation. Nearly 40% of the proteins differentially accumulated by the lack of CLPC2 were VC-responsive. Notably, 35S promoter-driven CLPC2 expression promoted changes in the proteome similar to those promoted by the lack of CLPC1. Collectively, our findings highlighted contrasting functional and molecular specificities for CLPC1 and CLPC2, and provided strong evidence that CLPC2 plays specific roles in CLP complex-mediated regulation of plant growth, photosynthesis, embryogenesis, postgerminative seedling establishment and microbial VC responsiveness.

plant biology↗

bioRxiv2025

Assembly of two functionally-distinct protein import complexes in the outer membrane of plant chloroplasts

The TOC translocon delivers thousands of nucleus-encoded proteins to chloroplasts and related non-photosynthetic plastids. It comprises the {beta}-barrel channel, Toc75, and multiple isoforms of receptor GTPases, Toc33 and Toc159. However, exactly how TOC complexes are assembled in different plastid types is unknown. Here, we present detailed characterization of two distinct TOC complexes, TOC-P and TOC-N, from photosynthetic chloroplasts and non-photosynthetic plastids, respectively. The assembled complexes are distinguished by having different sets of receptors, but both possess Toc75 which we identify as a central hub in TOC biogenesis: assembly is driven by TOC75 expression, with the Toc33 and Toc159 being added sequentially thereafter. Integrative structural analysis revealed a modular architecture for TOC-P comprising a cytosolic GTPase receptor module linked flexibly to a membrane {beta}-barrel channel module. TOC-N has a similar overall architecture, albeit with some clear differences that likely account for observed functional differences related to client specificity.

cell biology↗

bioRxiv2022

Chloroplast protein import determines plant proteostasis and retrograde signaling

Proteins containing polyglutamine (polyQ) repeats are prone to aggregation and can lead to distinct human pathologies. For instance, Huntingtons disease is caused by an abnormal expansion of the polyQ stretch (> Q35) of Huntingtin (HTT) protein. However, plants express hundreds of proteins containing polyQ regions, but no pathologies arising from these factors have been reported to date. Here, we ask how plants maintain the proteostasis of polyQ-containing proteins, which are intrinsically enriched in the plant proteomes. To this end, we overexpressed an aggregation-prone fragment of human HTT (Q69) in plant cells. In contrast to invertebrate and mammalian transgenic models, we find that Arabidopsis thaliana plants suppress Q69 aggregation. This elevated proteostasis ability is mediated through the import and degradation of Q69 in chloroplasts. Conversely, inhibition of chloroplast protein import either genetically or pharmacologically reduces the capacity of plant cells to prevent Q69 aggregation. We find that Q69 interacts with the chloroplast stromal processing peptidase (SPP). Notably, expression of synthetic Arabidopsis SPP is sufficient to suppress aggregation of polyQ-expanded HTT in human cells. Beyond ectopically expressed Q69-HTT, endogenous polyQ-containing proteins also aggregate in Arabidopsis upon inhibition of chloroplast import. Among them, the plastid casein kinase 2 (pCK2), which contains a polyQ region next to the chloroplast targeting sequence motif, can also be localized into the nucleus. Upon inhibition of chloroplast import, pCK2 accumulates at higher levels in the nucleus and forms diamond-shaped amyloid-like fibrils surrounding the chloroplasts. These results indicate that the differential conformation and redistribution of pCK2 to the nucleus depends on chloroplast import efficiency, providing a role of polyQ repeats in chloroplast to nucleus communication (i.e. retrograde signaling). Together, our findings establish chloroplast protein import and proteases as determinants of polyQ proteostasis, with important implications for plant biology that can also lead to therapeutic approaches for human diseases that involve protein aggregation.

plant biology↗