Drought is a major constraint for plant growth and crop productivity that is receiving an increased attention due to global climate changes. Chloroplasts act as environmental sensors, however, only partial information is available on stress-induced mechanisms within plastids. Here, we investigated the chloroplast response to a severe drought treatment and a subsequent recovery cycle in tomato through physiological, metabolite and proteomic analyses. Under stress conditions, tomato plants showed stunted growth, and elevated levels of proline, abscisic acid (ABA) and late embryogenesis abundant gene transcript. Proteomics revealed that water deficit deeply affects chloroplast protein repertoire (31 differentially represented components), mainly involving energy-related functional species. Following the rewatering cycle, physiological parameters and metabolite levels indicated a recovery of tomato plant functions, while proteomics revealed a still ongoing adjustment of the chloroplast protein repertoire, which was even wider than during the drought phase (54 components differentially represented). Changes in gene expression of candidate genes and accumulation of ABA suggested the activation under stress of a specific chloroplast-to-nucleus (retrograde) signaling pathway and interconnection with the ABA-dependent network. Our results give an original overview on the role of chloroplast as enviromental sensor by both coordinating the expression of nuclear-encoded plastid-localised proteins and mediating plant stress response. Although our data suggest the activation of a specific retrograde signaling pathway and interconnection with ABA signaling network in tomato, the involvement and fine regulation of such pathway need to be further investigated through the development and characterization of ad hoc designed plant mutants.

Cellular senescence is a permanent cell cycle arrest triggered by different stimuli. We recently identified up‐regulation of microRNA (miR)‐494 as a component of the genetic program leading to senescence of human diploid IMR90 fibroblasts. Here, we used 2‐dimensional differential gel electrophoresis (2D‐DIGE) coupled to mass spectrometry to profile protein expression changes induced by adoptive overexpression of miR‐494 in IMR90 cells. miR‐494 induced robust perturbation of the IMR90 proteome by significantly ( P <0.05) down‐regulating a number of proteins. Combination of mass spectrometry‐based identification of down‐regulated proteins and bioinformatic prediction of the miR‐494 binding sites on the relevant mRNAs identified 26 potential targets of miR‐494. Among them, computational analysis identified 7 potential evolution‐conserved miR‐494 targets. Functional miR‐494 binding sites were confirmed in 3'‐untranslated regions (UTRs) of 4 of them [heterogeneous nuclear ribonucleoprotein A3 (hnRNPA3), protein disulfide isomerase A3 (PDIA3), UV excision repair protein RAD23 homolog B (RAD23B), and synaptotagmin‐binding cytoplasmic RNA‐interacting protein (SYNCRIP)/heterogeneous nuclear ribonucleoprotein Q (hnRNPQ)]. Their reduced expression correlated with miR‐494 up‐regulation in senescent cells. RNA interference‐mediated knockdown of hnRNPA3 and, to a lesser extent, RAD23B mirrored the senescent phenotype induced by miR‐494 overexpression, blunting cell proliferation and causing up‐regulation of SA‐β‐galactosidase and DNA damage. Ectopic expression of hnRNPA3 or RAD23B slowed the appearance of the senescent phenotype induced by miR‐494. Overall, these findings identify novel miR‐494 direct targets that are involved in cellular senescence.—Comegna, M., Succoio, M., Napolitano, M., Vitale, M., D'Ambrosio, C., Scaloni, A., Passaro, F., Zambrano, N., Cimino, F., Faraonio, R. Identification of miR‐494 direct targets involved in senescence of human diploid fibroblasts. FASEB J. 28, 3720–3733 (2014). www.fasebj.org