Abstract
The stress-induced attachment of small ubiquitin-like modifier (SUMO) to a diverse collection of nuclear proteins regulating chromatin architecture, transcription, and RNA biology has been implicated in protecting plants and animals against numerous environmental challenges. In order to better understand stress-induced SUMOylation, we combined stringent purification of SUMO conjugates with isobaric tag for relative and absolute quantification mass spectrometry and an advanced method to adjust for sample-to-sample variation so as to study quantitatively the SUMOylation dynamics of intact Arabidopsis seedlings subjected to stress. Inspection of 172 SUMO substrates during and after heat shock (37 °C) revealed that stress mostly increases the abundance of existing conjugates, as opposed to modifying new targets. Some of the most robustly up-regulated targets participate in RNA processing and turnover and RNA-directed DNA modification, thus implicating SUMO as a regulator of the transcriptome during stress. Many of these targets were also strongly SUMOylated during ethanol and oxidative stress, suggesting that their modification is crucial for general stress tolerance. Collectively, our quantitative data emphasize the importance of SUMO to RNA-related processes protecting plants from adverse environments.
Highlights
The ability of cellular organisms to cope with environmental challenges requires the detection and immediate initiation of defense responses designed to mitigate the damage inflicted and enhance the organism’s ability to tolerate future insults
Quantifying Global Changes in SUMOylation during Heat Stress—Prior non-quantitative studies documented the changes in SUMOylation during and after a heat stress of Arabidopsis seedlings and revealed a dynamic time course in which heat (37 °C) rapidly induces the accumulation of SUMO1/2 conjugates, and this increase is followed by a slower decline after the seedlings are returned to their normal growth temperature (24 °C) [7, 8] (Fig. 1A)
Development of a Quantitative Proteomic Strategy to Monitor Changes in SUMOylation—Prior proteomic studies revealed that the high molecular mass collection of SUMO1/2 conjugates in Arabidopsis seedlings before and after heat shock includes over 350 nuclear-enriched substrates, with evidence that some are strongly SUMOylated by heat stress, whereas others are only weakly affected or even deSUMOylated [41]
Summary
Plant Materials and Quantitative Immunoblot Analysis—All experiments were conducted with the Arabidopsis thaliana Columbia-0 ecotype grown at 24 °C in liquid culture under continuous light [7, 41]. To search for possible SUMO and Ub footprints (QTGG or GG isopeptide linked to K, respectively [41, 50]), the database search was modified to include (i) a cyclized N-terminal glutamine (mass shift of 326.12 Da) for SUMO footprints, (ii) a non-cyclized version of the SUMO footprint with an N-terminal iTRAQ tag (total mass shift of 487.25 Da), and (iii) the Ub footprint (GG) with an N-terminal iTRAQ tag (mass shift of 258.25 Da) These data were filtered using a 1% peptide FDR. To validate the iTRAQ quantification, samples were generated by mixing and digesting equal volumes of purified SUMO conjugates obtained at each of four heat stress time points (t ϭ 0, 0.5, 1.5, and 4 h) together in solution with trypsin, and aliquots were separately labeled with each of four iTRAQ reagents.
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