A genetic approach reveals different modes of action of prefoldins

[EN] The prefoldin complex (PFDc) was identified in humans as a co-chaperone of the cytosolic chaperonin T-COMPLEX PROTEIN RING COMPLEX (TRiC)/CHAPERONIN CONTAINING TCP-1 (CCT). PFDc is conserved in eukaryotes and is composed of subunits PFD1-6, and PFDc-TRiC/CCT folds actin and tubulins. PFDs also participate in a wide range of cellular processes, both in the cytoplasm and in the nucleus, and their malfunction causes developmental alterations and disease in animals and altered growth and environmental responses in yeast and plants. Genetic analyses in yeast indicate that not all of their functions require the canonical complex. The lack of systematic genetic analyses in plants and animals, however, makes it difficult to discern whether PFDs participate in a process as the canonical complex or in alternative configurations, which is necessary to understand their mode of action. To tackle this question, and on the premise that the canonical complex cannot be formed if one subunit is missing, we generated an Arabidopsis (Arabidopsis thaliana) mutant deficient in the six PFDs and compared various growth and environmental responses with those of the individual mutants. In this way, we demonstrate that the PFDc is required for seed germination, to delay flowering, or to respond to high salt stress or low temperature, whereas at least two PFDs redundantly attenuate the response to osmotic stress. A coexpression analysis of differentially expressed genes in the sextuple mutant identified several transcription factors, including ABA INSENSITIVE 5 (ABI5) and PHYTOCHROME-INTERACTING FACTOR 4, acting downstream of PFDs. Furthermore, the transcriptomic analysis allowed assigning additional roles for PFDs, for instance, in response to higher temperature.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness and "Agencia Estatal de Investigacion"/FEDER/European Union (BIO2013-43184-P to D.A. and M.A.B., and BIO2016-79133-P and PID2019-109925GB-I00 to D.A.). N.B.-T., A.S.-M., and A.P.-A. were recipient of Ministerio de Economia y Competitividad (BES-2014-068868), EU MSCA-IF (H2020-MSCA-IF-2016746396) and Ministerio de Educacion (FPU17/05186) fellowships, respectively.Esteve-Bruna, D.; Blanco-Touriñán, N.; Serrano-Mislata, A.; Esquinas-Ariza, RM.; Resentini, F.; Forment Millet, JJ.; Carrasco-López, C.... (2021). A genetic approach reveals different modes of action of prefoldins. Plant Physiology. 187(3):1534-1550. https://doi.org/10.1093/plphys/kiab348S15341550187

Análisis de la contribución de las diferentes subunidades del complejo prefoldina al desarrollo y a la respuesta al ambiente en Arabidopsis

[ES] El complejo de prefoldina se identificó originalmente en humanos como co-chaperona que ayuda a la CCT de chaperonina citosólica en el plegamiento de actina y tubulinas. Es un complejo heteromérico formado por seis proteínas pequeñas no relacionadas, prefoldina 1-6, y se conserva en eucariotas. En Arabidopsis, los mutantes simpoles de dfp presentan defectos graves en la organización de los microtúbulos, con consecuencias directas en la expansión celular de los hipocotilos y la raíz primaria, y en algunos casos, también son defectuosos en la adaptación al estrés ambiental. Sin embargo, queda por determinar en qué medida las diferentes prefoldinas, individualmente o en complejo, contribuyen al desarrollo de la planta y a las respuestas al estrés. El propósito de esta tesis de Master es la caracterización funcional de todos los mutantes de dfp simples, quíntuples y sextuples para tratar de asignar funciones específicas y / o determinar la contribución de cada subunidad prefoldina a diferentes procesos de desarrollo e.g. crecimiento de la raíz, tiempo de floración, tamaño y forma de roseta, etc., y respuesta al estrés, p. ej. Sal y osmótica. Finalmente, los análisis morfológicos y fisiológicos se complementarán con dos enfoques moleculares: (i) una secuencia de ARN para analizar el transcriptoma del mutante sextuple, y (ii) inmunoblots y análisis confocales en mutantes dfp para determinar defectos en la acumulación de tubulina y la organización de los microtúbulos .[EN] The prefoldin complex was originally identified in humans as a co-chaperone helping the cytosolic chaperonin CCT in the folding of actin and tubulins. It is a highly conserved heterohexameric complex composed of subunits PFD1 to 6. In Arabidopsis, single pfd mutants display severe defects in microtubule organization, with direct consequences in cellular expansion of hypocotyls and primary root, and in some cases, are defective in the adaptation to environmental stress as well. However, it remains to be determined the extent to which the different prefoldins, individually or in complex, contribute to these responses. In this master¿s thesis, we have confirmed that PFD1 and PFD2 act as canonical PFDs, due to the microtubule organization defects observed in the previously uncharacterized pfd1 and pfd2 mutants. Importantly, we have prepared a mutant defective in the six PFD genes, pfdx6. A transcriptomic analysis of the sextuple mutant revealed novel roles for PFD in germination, lateral root development and in response to hormones (auxin and ABA). Our phenotypic analyses of the singles pfd and the pfdx6 mutants has allowed us to discern between processes that depend on the activity of the whole complex and those that are controlled redundantly by more than one PFD subunit. Our results reveal that the PFD complex, although it is not essential for the viability of the plant, it has a relevant role in development processes as well as in the adaptation to the environment.Esquinas Ariza, RM. (2020). Analysis of the contribution of the different prefoldin complex subunits to Arabidopsis development and response. http://hdl.handle.net/10251/136725TFG